Claims:

1. A method of treatment of a tumor in a patient in need of such
treatment, said tumor comprising cancer cells characterized by at least
one of butyrylcholinesterase expression and androgen binding affinity,
said method comprising administering to said patient a therapeutically
effective amount of at least one compound of the formula: ##STR00012##
wherein Ra represents OH or: ##STR00013## X represents H, F, Cl,
or a C1-C8 alkyl, or C1-C8 alkoxy group; Y represents
H, C1-C8 alkyl, C5-C14 aryl, or a C5-C14
aryloxy group; Z represents H, C1-C8 alkyl, C5-C14
aryl, or a C5-C14 aryloxy group; R represents halogen,
radiohalogen, or a C1-C8 alkyl, C5-C14 aryl,
C1-C8 alkylthio, C1-C8 alkoxy, C2-C6
alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, or
Sn(C1-C4 alkyl)3 group; Rb represents halogen,
radiohalogen, or a C1-C6 alkoxy or C1-C8 alkanoate
group, an androgen receptor binding ligand linked to the said compound
via a cleavable linking moiety, or: ##STR00014## any of said alkyl,
alkenyl, alkynyl, alkylthio, alkoxy and cycloalkyl group being optionally
substituted by at least one halogen, OH, SH, NH2, C1-C4
monoalkylamino, C1-C4 dialkylamino, COOH, CN, NO2,
C1-C4 alkyl, C1-C4 alkoxy or phenyl group, any of
said aryl, aryloxy, and phenyl group being optionally substituted by at
least one halogen, OH, SH, NH2, C1-C4 monoalkylamino,
C1-C4 dialkylamino, COOH, CN, NO2, C1-C4 alkyl
or C1-C4 alkoxy group; said radiohalogen represents 123I,
124I, 125I, 131I, 211At, 18F, 76Br,
77Br, or 80mBr; stereoisomeric forms and pharmaceutically
acceptable salts of said at least one compound; with the proviso that at
least one of the Ra and Rb substitutents represents:
##STR00015## and the wavy line indicating the point of attachment to the
ribose moiety.

2. The method of claim 1, wherein said patient is administered at least
one compound of the formula: ##STR00016## wherein X represents H, F,
Cl, or a C1-C4 alkyl, or C1-C4 alkoxy group; Y
represents H or a C1-C4 alkyl group; Z represents H or a
C1-C4 alkyl group; R represents halogen, radiohalogen, or a
C1-C4 alkyl, C1-C4 alkoxy or phenyl group; Rb
represents halogen, radiohalogen, OH or a C1-C4 alkoxy group,
or an androgen receptor binding ligand linked to the said compound via a
cleavable linking moiety; any of said alkyl, alkoxy and phenyl group
being optionally substituted by at least one halogen, OH, SH, NH2,
C1-C4 monoalkylamino, C1-C4 dialkylamino, COOH, CN,
NO2, C1-C4 alkyl or C1-C4 alkoxy; and said
radiohalogen represents 123I, 124I, 125I, 131I,
211At, 18F, 76Br, 77Br, or 80mBr; and
stereoisomeric forms and pharmaceutically acceptable salts of said at
least one compound.

3. The method of claim 1, wherein the androgen receptor binding ligand is
selected from the group consisting of an androgen receptor agonist and an
androgen receptor antagonist.

4. The method of claim 3, wherein the androgen receptor agonist is
selected from the group consisting of 4-dihydrotestosterone (DHT),
testosterone, mibolerone, methyltrienolone, and methyltestosterone.

5. The method of claim 3, wherein the androgen receptor antagonist is
selected from the group consisting of hydroxyflutamide, flutamide,
cyproterone acetate, spironolactone, ketoconazole, and finasteride.

6. The method of claim 1, wherein said patient is administered a compound
selected from the group consisting of: ##STR00017##

7. The method of claim 6, wherein said compound (A), (B), (C) or (D) is
administered in the form of the fast isomer, the slow isomer, or a
mixture thereof.

8. The method of claim 1, wherein said compound is administered in the
form of the slow isomer thereof.

9. The method of claim 1, wherein the cancer cells are selected from the
group consisting of ovarian, glioma, colorectal, breast, prostate,
meningioma, head and neck, or pancreatic cancer cells.

10. The method of claim 1, wherein said at least one compound is
administered by a method selected from the group consisting of
intravenous, intraperitoneal, and intratumoral administration.

11. The method of claim 10, wherein said at least one compound is
administered periodically for a term of years.

12. The method of claim 11, wherein said at least one compound is
administered daily.

13. A method of diagnosing a patient for the presence of a tumor, said
tumor comprising cancer cells characterized by at least one of
butyrylcholinesterase expression and androgen binding affinity, said
method comprising administering to said patient an effective amount for
diagnostic imaging of a compound of the formula: ##STR00018## wherein
Ra represents OH or: ##STR00019## X represents H, F, Cl, or a
C1-C8 alkyl, or C1-C8 alkoxy group; Y represents H,
C1-C8 alkyl, C5-C14 aryl, or a C5-C14
aryloxy group; Z represents H, C1-C8 alkyl, C5-C14
aryl, or a C5-C14 aryloxy group; R represents halogen,
radiohalogen, or a C1-C8 alkyl, C5-C14 aryl,
C1-C8 alkylthio, C1-C8 alkoxy, C2-C6
alkenyl, C2-C6 alkynyl, C3-C12 cycloalkyl, or
Sn(C1-C4 alkyl)3 group; Rb represents halogen,
radiohalogen, or a C1-C6 alkoxy or C1-C8 alkanoate
group, an androgen receptor binding ligand linked to the said compound
via a cleavable linking moiety, or: ##STR00020## any of said alkyl,
alkenyl, alkynyl, alkylthio, alkoxy and cycloalkyl group being optionally
substituted by at least one halogen, OH, SH, NH2, C1-C4
monoalkylamino, C1-C4 dialkylamino, COOH, CN, NO2,
C1-C4 alkyl, C1-C4 alkoxy or phenyl group, any of
said aryl, aryloxy, and phenyl group being optionally substituted by at
least one halogen, OH, SH, NH2, C1-C4 monoalkylamino,
C1-C4 dialkylamino, COOH, CN, NO2, C1-C4 alkyl
or C1-C4 alkoxy group; said radiohalogen represents 123I,
124I, 125I, 131I, 211At, 18F, 76Br,
77Br, or 80mBr; stereoisomeric forms and pharmaceutically
acceptable salts of said at least one compound; with the proviso that at
least one of the Ra and Rb substitutents represents:
##STR00021## the wavy line indicating the point of attachment to the
ribose moiety; and performing imaging to diagnose the presence of said
tumor.

14. The method of claim 13, wherein the androgen receptor binding ligand
is selected from the group consisting of an androgen receptor agonist and
an androgen receptor antagonist.

15. The method of claim 14, wherein the androgen receptor agonist is
selected from the group consisting of 4-dihydrotestosterone (DHT),
testosterone, mibolerone, methyltrienolone, and methyltestosterone.

16. The method of claim 14, wherein the androgen receptor antagonist is
selected from the group consisting of hydroxyflutamide, flutamide,
cyproterone acetate, spironolactone, ketoconazole, and finasteride.

17. The method of claim 13, wherein said imaging is selected from the
group consisting of scintigraphic imaging and magnetic resonance
spectroscopy.

18. The method of claim 17, wherein said scinintigraphic imaging is
selected from the group consisting of positron emission tomography and
single photon emission computed tomography.

19. The method of claim 13, wherein the cancer cells are selected from
the group consisting of ovarian, glioma, colorectal, breast, prostate,
meningioma, head and neck, or pancreatic cancer cells.

20. The method of claim 13, wherein said patient is administered a
compound of the formula: ##STR00022##

21. A method for monitoring tumor activity in a subject, said method
comprising administering an effective amount for diagnostic imaging of a
compound of the formula: ##STR00023## wherein Ra represents OH or:
##STR00024## X represents H, F, Cl, or a C1-C8 alkyl, or
C1-C8 alkoxy group; Y represents H, C1-C8 alkyl,
C5-C14 aryl, or a C5-C14 aryloxy group; Z represents
H, C1-C8 alkyl, C5-C14 aryl, or a C5-C14
aryloxy group; R represents halogen, radiohalogen, or a C1-C8
alkyl, C5-C14 aryl, C1-C8 alkylthio, C1-C8
alkoxy, C2-C6 alkenyl, C2-C6 alkynyl,
C3-C12 cycloalkyl, or Sn(C1-C4 alkyl)3 group;
Rb represents halogen, radiohalogen, or a C1-C6 alkoxy or
C1-C8 alkanoate group, an androgen receptor binding ligand
linked to the said compound via a cleavable linking moiety, or:
##STR00025## any of said alkyl, alkenyl, alkynyl, alkylthio, alkoxy and
cycloalkyl group being optionally substituted by at least one halogen,
OH, SH, NH2, C1-C4 monoalkylamino, C1-C4
dialkylamino, COOH, CN, NO2, C1-C4 alkyl, C1-C4
alkoxy or phenyl group, any of said aryl, aryloxy, and phenyl group being
optionally substituted by at least one halogen, OH, SH, NH2,
C1-C4 monoalkylamino, C1-C4 dialkylamino, COOH, CN,
NO2, C1-C4 alkyl or C1-C4 alkoxy group; said
radiohalogen represents 123I, 124I, 125I, 131I,
211At, 18F, 76Br, 77Br, or 80mBr; stereoisomeric
forms and pharmaceutically acceptable salts of said at least one
compound; with the proviso that at least one of the Ra and Rb
substitutents represents: ##STR00026## the wavy line indicating the
point of attachment to the ribose moiety; obtaining an image of said
tumor to establish a baseline tumor size in said subject; readministering
said compound to said subject; and obtaining at least one other image of
said tumor producing a result which is indicative of the tumor activity
in said patient.

22. The method of claim 21, wherein the androgen receptor binding ligand
is selected from the group consisting of an androgen receptor agonist and
an androgen receptor antagonist.

23. The method of claim 22, wherein the androgen receptor agonist is
selected from the group consisting of 4-dihydrotestosterone (DHT),
testosterone, mibolerone, methyltrienolone, and methyltestosterone.

24. The method of claim 22, wherein the androgen receptor antagonist is
selected from the group consisting of hydroxyflutamide, flutamide,
cyproterone acetate, spironolactone, ketoconazole, and finasteride.

25. The method of claim 21, wherein said subject undergoes therapy for
treatment of said tumor between establishment of said baseline tumor size
and obtaining said at least one other image of said tumor.

26. The method of claim 21, wherein said imaging is selected from the
group consisting of scintigraphic imaging and magnetic resonance
spectroscopy.

27. The method of claim 26, wherein said scintigraphic imaging is
selected from the group consisting of positron emission tomography and
single photon emission computed tomography.

28. The method of claim 25, wherein said treatment is at least one of
surgical excision, radiation therapy and chemotherapy.

[0003] The present invention relates generally to therapeutic and
diagnostic applications of radiolabeled synthetic compounds, which are
effective to (1) kill cancer cells undergoing DNA replication or repair
by incorporation into the growing DNA strand resulting in DNA double
strand breaks, (2) specifically target two membrane proteins expressed in
cancer cells and implicated in tumorogenesis, butyrylcholinesterase
(BChE) and the androgen receptor (AR). The compounds of the invention are
taken up selectively by malignant tumor cells and are incorporated into
the nucleus of such cells, where they produce a cytotoxic effect and/or
are detectable via nuclear medicine imaging techniques.

[0004] The main treatments for ovarian, breast, prostate and many other
cancers are surgery, chemotherapy and radiation therapy. In some cases a
combination of two or more of these treatments is recommended. Typically,
clinical trials for advanced carcinomas use combination chemotherapy
based on established anti-cancer agents. For example, there are numerous
active clinical trials (Phase I) dealing with recurrent and progressive
ovarian carcinoma that rely on existing drugs such as paclitaxel,
carboplatin, cisplatin, floxouridine and similar drugs in a combination
chemotherapy. Many of these include autologous stem cell support to
combat the side effects brought on by the administration of these drugs.
Newer drugs include matrix metalloproteinase inhibitors, vaccines, and
antibodies.

[0005] The prognosis is relatively poor for patients diagnosed with
high-grade gliomas with glioblastoma multiforme patients rarely surviving
beyond 12 months. The standard treatments for brain gliomas entail a
multifaceted approach providing radiation, surgery, and chemotherapy.
Many chemotherapeutic approaches are ineffective, due to the inability of
most chemotherapeutics to cross the blood-brain barrier, and/or are
overly toxic. Moreover, there is little ability to prevent recurrence
following surgical resection. Palliative therapies for glioma besides
radiation therapy and surgical resection include Avastin (bevacizumab)
and temozolomide in combination with radiation therapy.

[0006] Many of the currently available front-line and salvage agents used
in cancer therapy are associated with cumulative and/or irreversible
toxicities that pose challenges for long-term treatment planning. The
irreversible effects associated with some of these therapies include
development of multidrug resistance, neurotoxicity, and nephrotoxicity.
All of these diminish the probability of improved responses when multiple
treatments are needed to keep the cancer under control.

[0007] It has previously been proposed to use targeted cytotoxic
radioisotopes for the treatment and diagnosis of cancer. One of the
intended benefits of targeted therapy is to diminish the incidence and
severity of side effects by confining toxic exposure, more or less, to
the disease site. Certain radioisotopes, particularly Auger
electron-emitting isotopes, such as 123I and 125I are known to
be very toxic to viable cells, but only if they are localized within the
nucleus of the cell (Warters et al., Curr. Top. Stop Rad. Res., 12:389
(1977)). It has been reported that 5'-iodo-2'-deoxyuridine (IUdR), when
labeled with the Auger electron emitter 123I or 125I exhibits
substantial toxicity in mammalian cells in vitro (Makrigiorgos et al.,
Radiat. Res., 118:532-44 (1989)) and produces a therapeutic effect in
animal tumor models (Baranowska-Kortylewicz et al., Int. J. Radiat.
Oncol. Biol. Phys., 21:1541-51 (1991)). Furthermore, radiolabeled IUdR
has been found to enable scintigraphic detection of animal and human
tumors (Baranowska-Kortylewicz, supra). See also U.S. Pat. Nos. 5,094,835
and 5,308,605.

[0008] Considerable effort has been devoted to developing antibodies for
the targeted delivery of therapeutic and diagnostic agents. However,
antibodies themselves have not been capable of reaching the cell nucleus
in effective amounts. Most such antibodies react with the cell surface,
and are gradually internalized, routed to lysosomes and degraded
(Kyriakos et al., Cancer Res., 52:835 (1992)). Degradation products,
including any radioisotopes attached thereto, then gradually leave the
cell by crossing the lysosomal membrane and then the cell membrane.
Although a conventional radioisotope label on an antibody degradation
product can theoretically pass through the nuclear membrane and deliver
some radioactivity to the nucleus (Woo et al., WO 90/03799) actual
observations show that the amount is limited, and in any event, is
insufficient to have a toxic effect on tumor cells.

[0009] Protein and polypeptide hormones and growth factors, particularly
those having cell surface receptors, may be directly radiolabeled and
used to target a tumor cell. As in the case of targeting radiolabeled
antibodies, however, radioisotopes bound to amino acid residues of
hormones, growth factors and the like exit from the cell after
catabolism, and do not appreciably bind to nuclear material.

[0010] U.S. Pat. No. 7,220,730, which is commonly owned with this
application, relates to cancer specific radiolabeled conjugates useful as
both therapeutic and diagnostic agents in the treatment of cancer. The
radiolabeled conjugates incorporate a component that is effective to
target tumor cells, which cells selectively take up and degrade the
conjugate. The unmasked radioisotopic moiety is then delivered to the
nucleus and incorporated into the nuclear material so as to produce a
cytotoxic effect and/or render the cell detectable to nuclear medicine
imaging.

[0011] Despite the many advances in the field of cancer therapy and
diagnosis, a need still exists for innovative cancer treatment and
diagnostic methods, which can be safely applied in a repetitive,
long-term regimen, without the side effects produced by existing
treatments. This is especially true with respect to therapeutic
modalities for cancers that have high instances of relapse.

SUMMARY OF THE INVENTION

[0012] The above-noted need is satisfied by the compounds of the present
invention which are capable of binding to and being selectively taken up
and degraded by a tumor composed of cancer cells having certain markers,
and thereby delivering to the cell nucleus a radioisotope capable of
being incorporated into the nuclear material, so as to produce a
cytotoxic effect and/or to render the tumor cell detectable by nuclear
medicine imaging. The compounds of the invention can be safely
administered in long-term cancer treatments, without producing
significant adverse health effects.

[0013] In accordance with one aspect of the present invention, there is
provided a method of treatment of a tumor comprising cancer cells in a
patient by administrating a therapeutically effective amount of a
compound of the formula:

[0021] with the proviso that at least one of the Ra and Rb
substitutents represents:

##STR00004##

and

[0022] the wavy line indicating the point of attachment to the ribose
moiety.

[0023] In another aspect of the invention, the compounds of this invention
can be administered as either fast or slow eluting isomers or a mixture
thereof, as will be explained below in further detail.

[0024] The compounds of this invention can be used to eradicate residual
cancer cells, e.g. in relapsing cancers, with minimal, if any, damage to
normal tissues or to tissues in and around the treatment site. The method
of the present invention may be applied for treating and diagnosing
cancers comprising cells characterized by at least one of BChE expression
and androgen binding affinity, including, without limitation, ovarian,
breast, prostate, head and neck, pancreatic, glioma, colorectal, and
meningioma malignant tumors.

[0025] The compounds of the present invention have been designed so as to
take advantage of three characteristics of many relapsing cancers, i.e.
(1) relapsed/advance cancers have a large portion of rapidly growing and
dividing cells (i.e. a large S-phase fraction); (2) AR is expressed in
practically all prostate cancer (primary and metastatic), ovarian cancer
(>90% positive for AR regardless of the tumor site) and breast cancer
(even when estrogen receptor (ER)-negative and progesterone receptor
(PR)-negative, breast cancer cells express AR), glioma, head and neck
cancer, colorectal cancer, and meningiomas; and (3) BChE is expressed in
a variety of cancer types allowing for malignant tumor targeting and
avoidance of impacting surrounding healthy tissue thereby enabling the
compounds of the invention to be used to treat non-resectable malignant
tumors.

[0026] The compounds of formula (I), above, can also be used to advantage
for diagnosis of malignant tumors. The method comprises administering to
a patient a diagnostically effective amount of labeled compound of
formula (I), and then imaging the tumor by scintigraphic imaging or
magnetic resonance spectroscopy. This method can also be adapted for use
in monitoring tumor activity in a subject.

[0027] Also in accordance with the invention there is provided a compound
of the formula:

[0033] The cytotoxic effects of the methods of the invention are induced
only when one or more of the compounds described herein are incorporated
into the DNA of rapidly dividing tumor cells. This dependence of
radiotoxicity on the participation of the compound in DNA synthesis, in
combination with relatively rapid pharmacokinetics, limits the exposure
of normal tissue to radiation. In other words, the compound(s) that
remain(s) in systemic circulation, or enter(s) normal tissue or organs,
is (are) essentially innocuous. Accordingly, the compounds of the
invention may be administered frequently and without appreciable adverse
effects.

[0034] A kit comprising a vessel containing a compound of formula (I),
above, and a pharmaceutically acceptable carrier medium is also provided.

[0043] FIG. 9 is a set of graphs comparing blood, tumor, and several
normal tissue clearance curves for 8b fast.

[0044] FIG. 10 is a set of graphs comparing blood, tumor, and several
normal tissue clearance curves for 8b slow.

[0045]FIG. 11 is a (A) graphical representation of the weights of solid
tumors and tumor cells recovered in ascites of OVCAR-3 bearing mice seven
weeks after the first dose of the radiolabeled diastereomeric mixture of
compounds 6b (6b mix), 7b (7b mix), or the parent compound 125IUdR,
and (B) graphical representation of the whole body radioactivity of the
mice two weeks after treatment.

[0046]FIG. 12 is a graphical representation of the retention of
radioactivity in solid tumor deposits and in the cancer cells in ascites
42 days after the administration of the radioactive compounds 6b mix, 7b
mix, and the parent compound 125IUdR.

[0048] FIG. 14 is a set of dose response curves for the and solid tumor
burden (A) and cancer cells in ascites fluid (B) in OVCAR-3-bearing
athymic mice treated with fractionated doses of 8b slow.

[0049] FIG. 15 is the summary of the hematological values in
OVCAR-3-bearing mice treated with fractionated doses of 8b slow.
Hemoglobin and hematocrit values in athymic mice bearing IP OVCAR-3 tumor
implants 48 days after treatment with the fractionated doses of 8b slow.

[0050]FIG. 16 is a graphical representation of the results of statistical
analyses of tumor burden and hematological parameters in the fractionated
therapy study. Bolded values indicate the statistically significant
differences, i.e. P<0.05.

[0051] FIG. 17 is a graphical representation of OVCAR-3 tumor weights
obtained during post-therapy necropsy. The left panel shows the weight of
solid tumor deposits. The right panel shows the weight of the cancer cell
pellet recovered with the peritoneal lavage. There is a demonstrated
dose-dependent response to 8b slow.

[0052] FIG. 18 is a graphical representation of in vitro kinetics of 7b
uptake in OVCAR-3 human adenocarcinoma cells. The average radioactive
concentration in each well was 0.74±0.04 μCi/mL (27.3±1.4
kBq/mL).

[0053] FIG. 19 is a graphical representation of in vitro kinetics of 7b
uptake in LS174T human colorectal cancer cells. The average radioactive
concentration in each well was 0.76±0.06 μCi/mL (28.2±2.2
kBq/mL).

[0054] FIG. 20 is a graphical representation of changes in the surviving
fraction of LS174T cells grown with 6b fast and 6b slow. Clonogenic assay
was performed on cells exposed to radioactive compounds for 4 h followed
by additional 24 h culture in nonradioactive media before the cell
harvest and plating at densities suitable for clonogenic assay.

[0055] FIG. 21 is a graphical representation of the surviving fraction of
LS174T colorectal adenocarcinoma cells treated with 7b fast and 7b slow
for 4 hours at a compound concentration of 5 μCi/mL (185 kBq/mL).
Cells were harvested immediately after the exposure to the compound and
re-plated at densities suitable for the clonogenic assay.

[0056] FIG. 22 is a graphical representation of the subcellular
distribution of 6b mix in OVCAR-3 cancer cells measured at 1 hour and 24
hours.

[0059] FIG. 25 is a graphical representation of cellular uptake and
subcellular distribution of 6b in U-87 human glioblastoma cells after 24
hour exposure to 6b as isolated fast and slow isomers.

[0060] FIG. 26 is a graphical representation of the subcellular
distribution and retention of 6b fast and 6b slow in DNA of U-87 human
glioblastoma cells.

[0061] FIG. 27 is a graphical representation of the
concentration-dependent uptake of 6b, as isolated fast and slow isomers,
by U-87 human glioblastoma cells.

DETAILED DESCRIPTION OF THE INVENTION

[0062] The compounds of formula (I), above, are composed of one component
which is effective for killing cancer cells undergoing rapid DNA
replication in addition to one or more specific targeting components
capable of targeting BChE and/or AR expressing malignant tumor cells.
Additionally, these compounds bind sex-hormone binding globulin (SHBG),
which increases their half-life in the serum and allows uptake of the
compounds in the cell via the SHBG receptor

[0063] Iodine-125, for example, damages DNA and efficiently kills cells
only when it is located in the cell nucleus near or within DNA. The use
of this radioisotope is beneficial because it is practically harmless
when present in extracellular spaces. Thus, these compounds, if used
alone or in combination therapies, will not increase the overall toxicity
of the primary treatment.

[0064] Thymidine analogs, such as IUdR, when radiolabeled with an Auger
emitter, are essentially innocuous outside the cell and ineffective at
killing cells inside the cytoplasm. IUdR may also be radiolabeled with
alpha- and beta-emitters. Unlike Auger electron emitters, these
radioisotopes are radiotoxic even when outside the cell. Such isotopes
would allow for the irradiation of neighboring cells, i.e., a bystander
effect, which is beneficial, particularly if AR, BChE, and/or SHBG
expression is not uniform. IUdR is, for the most part, taken up
selectively by dividing malignant tumor cells. These cells are located
within a niche of nondividing cells and the radioactive compound(s) can
be indefinitely retained within the nucleus of the cancer cell following
DNA incorporation. Nondividing cells will not incorporate radiolabeled
IUdR into their DNA and most of the radiolabeled IUdR that is not taken
up by cancerous cells will be catabolized/dehalogenated rapidly
(t1/2 measurable in minutes) and thus, will not incorporate in the
DNA of distant non-cancerous dividing cells.

[0065] Furthermore, since radiolabeled IUdR is a small molecule it will
not induce an immune response, which permits repeated injections,
continuous infusion, or similar modes of administration. In order to
provide cancer cell specificity and enhanced delivery, in certain
embodiments of this invention the radiolabeled thymidine analogs are
conjugated to a BChE selective ligand. In other embodiments of this
invention, the radiolabeled thymidine analogs are conjugated to an AR
specific ligand. Additionally, in other embodiments of this invention,
the radiolabeled thymidine analogs are conjugated to both a BChE and an
AR ligand.

[0066] Radiolabeled IUdR, in one embodiment of this invention, may be
chemically coupled to a cycloSaligenyl phosphotriester moiety having
binding affinity for BChE. BChE plays a role in tumorigenesis and is
expressed predominantly on the membrane and in the cytosol of many
malignant tumor cells. BChE genes are amplified, mutated, and/or
aberrantly expressed in a variety of human tumor types. BChE contains the
consensus peptide motif S/T-P-X-Z, which is found in many substrates of
cdc2-related protein kinases suggesting that phosphorylation by
cdc2-related kinases may be the molecular mechanism linking BChE to tumor
proliferation. Studies have also demonstrated BChE upregulation in brain
tumors (Rios, et al., Surg Neurol 55:106-12 (2001)), ovarian carcinomas
(Zakut et al., J. Clin. Invest. 86:900-908 (1990)), breast cancer
(Bernardi et al., Cancer Genetics and Cytogenetics 197:158-165 (2010)),
and glioma (Saez-Valero et al., 206:173-176 (1996)).

[0067] The term "differential butyrylcholinesterase expression" as used
herein, refers to at least one recognizable difference in protein or
nucleic acid expression. It may be a quantitatively measureable,
semi-quantitatively estimatable or qualitatively detectable difference in
cells, tissue, or bodily fluid protein expression. Thus, differentially
expressed butyrylcholinesterase may be strongly expressed in cells,
tissue, or bodily fluid in the normal state and less strongly expressed
or not expressed at a measureable level in the damaged state. Conversely,
it may be strongly expressed in cells, tissue, or bodily fluid in the
damaged state, and less strongly expressed or not expressed at all in the
normal state.

[0076] It should be appreciated that compounds of formula (I) and (Ia),
above, may have one or more asymmetric centers and thus exist as
stereoisomers, including diastereomers, with stereocenters named
according to the Cahn-Ingold-Prelog system (R/S designation of
stereocenters). Although the structural formulas set forth above are
represented without regard to stereochemistry, it is intended to include
all possible stereoisomers, which may be diastereomeric mixtures, as well
as resolved, substantially pure optically active and inactive forms, and
pharmaceutically acceptable salts thereof.

[0077] Stereoisomers of the compounds used in the practice of this
invention can be selectively synthesized or separated into pure,
optically-active or inactive form using conventional procedures known to
those skilled in the art of organic synthesis. For example, mixtures of
stereoisomers may be separated by standard techniques including, but not
limited to, resolution of diastereomeric forms, normal, reverse-phase,
and chiral chromatography, preferential salt formation,
recrystallization, and the like, or by asymmetric synthesis either from
enantiomerically or diastereomerically pure starting materials or by
deliberate synthesis of target enantiomers or diastereomers. All of the
various isomeric forms of the compounds of formulas (I) and (Ia), above,
are within the scope of this invention. Nonstereoselective syntheses
produce the diastereometric mixture of cycloSaligenyl-phosphotriesters.
Isomers may be separated by reverse phase HPLC and resolved according to
their retention time as the slow and the fast diastereomers, as described
in further detail below. The slow diastereomers are more potent
inhibitors of BChE in contrast to the fast diastereomers.

[0078] The phrase "enantiomeric excess" or "ee" is a measure, for a given
sample, of the excess of one enantiomer over a racemic sample of a chiral
compound and is expressed as a percentage. Enantiomeric excess is defined
as 100*(er-1)/(er+1), where "er" is the ratio of the more abundant
enantiomer to the less abundant enantiomer.

[0079] The phrase "diastereomeric excess" or "de" is a measure, for a
given sample, of the excess of one diastereomer over a sample having
equal amounts of diastereomers and is expressed as a percentage.
Diastereomeric excess is defined as 100*(dr-1)/(dr+1), where "dr" is the
ratio of a more abundant diastereomer to a less abundant diastereomer.
The term does not apply if more than two diastereomers are present in the
sample.

[0080] Preferably, where the "substantially pure" compound of formula (I),
above, is provided as a diastereomer, the diastereomer is present at an
diastereomeric excess of greater than or equal to about 80%, more
preferably, at an diastereomeric excess of greater than or equal to about
90%, more preferably still, at an diastereomeric excess of greater than
or equal to about 95%, more preferably still, at an diastereomeric excess
of greater than or equal to about 98%, most preferably, at an
diastereomeric excess of greater than or equal to about 99%.

[0081] As used herein, the term "alkyl" refers to saturated straight and
branched chain hydrocarbon radicals, having 1-8 and preferably 1-4 carbon
atoms. The term "alkenyl" is used to refer to unsaturated straight and
branched chain hydrocarbon radicals including at least one double bond,
and having 2-6. Such alkenyl radicals may be in trans (E) or cis (Z)
structural configurations. The term "alkynyl" is used herein to refer to
both straight and branched unsaturated hydrocarbon radicals including at
least one triple bond and having 2-6.

[0082] The term "cycloalkyl" as used herein refers to a saturated cyclic
hydrocarbon radical with one or more rings, having 3-12.

[0083] Any alkyl, alkenyl, alkynyl or cycloalkyl moiety of a compound
described herein may be substituted with one or more groups, such as
halogen, OH, SH, NH2, C1-C4 monoalkylamino,
C1-C4 dialkylamino, COOH, CN, NO2, C1-C4 alkyl
or C1-C4 alkoxy.

[0084] The term "aryl" as used herein refers to an aromatic hydrocarbon
radical composed of one or more rings and having 5 or 6-14 carbon atoms
and preferably 5 or 6-10 carbon atoms, such as phenyl, naphthyl,
biphenyl, fluorenyl, indanyl, or the like. Any aryl moiety of a compound
described herein may be substituted with one or more groups, such as
halogen, OH, SH, NH2, C1-C4 monoalkylamino,
C1-C8 dialkylamino, COOH, CN, NO2, C1-C4 alkyl
or C1-C4 alkoxy. The aryl moiety is preferably substituted or
unsubstituted phenyl.

[0085] The term "halogen" or "halo" as used herein refers to Fl, Cl, Br
and I.

[0086] The term "radiohalogen" as used herein refers to an isotopic form
of halogen that exhibits radioactivity. The radiohalogen is preferably
selected from the group consisting of 123I, 124I, 125I,
131I, 211At, 18F, 76Br, 77Br, and 80mBr.

[0087] The term "alkoxy" refers to alkyl-O--, in which alkyl is as defined
above.

[0088] The term "alkylthio" refers to alkyl-S--, in which alkyl is as
defined above.

[0089] The term "carboxy" refers to the moiety --C(═O)OH.

[0090] The term "aryloxy" refers to the moiety --O-aryl, in which aryl is
defined above.

[0091] The term "alkanoate" refers to the moiety --O--C(═O)-alkyl, in
which alkyl is as defined as above.

[0092] The term "monoalkylamino" refers to the moiety --NH(alkyl), in
which alkyl is as defined as above.

[0093] The term "dialkylamino" refers to the moiety N(alkyl)2, in
which alkyl is as defined as above.

[0094] The cleavable linking moiety, L, can be a diester or a phosphate.
The diester moiety may be represented as
--O--C(═O)--(CH2)n--C(═O)--O--, wherein n=2-10. The
preferred cleavable linking moiety is a succinate moiety.

[0095] The term "androgen receptor binding ligand", is defined as an
androgen receptor agonist or an androgen receptor antagonist.

[0096] The androgen receptor agonists that may be used in accordance with
the present invention includes, without limitation, 4-dihydrotestosterone
(DHT), testosterone, mibolerone, methyltrienolone, and
methyltestosterone. The androgen receptor antagonists that may be used in
accordance with the present invention includes, without limitation,
hydroxyflutamide, flutamide, cyproterone acetate, spironolactone,
ketoconazole, and finasteride. In accordance with the present method of
the invention, synthetic modification of known androgen receptor agonists
and antagonists, to allow for linkage to the compounds used in the method
of the invention, would be well understood by a person having ordinary
skill in synthetic organic chemistry. The preferred ligand is DHT bound
through its hydroxyl substituent.

[0097] Based on BChE inhibition studies (IC50), structure-activity
relationships (SAR) have elucidated key structural features providing
enhanced BChE selectivity and inhibition. By appropriate selection of the
X substituent of the compound of formula (Ia), above, the rate of
hydrolysis of the cycloSal-phospho-triester moiety can be varied. For
example, the possibilities for X could include hydrogen, methyl, chloro,
fluoro, or methoxy radicals. Increased BChE inhibitory activity and
enhanced selectivity for BChE among slow/fast diastereomers at the
phosphorus atom is achieved through selection of a tert-butyl group on
the cycloSal-phospho-triester phenyl ring as the Y and Z substituent.
There is also increased BChE inhibitory activity and enhanced selectivity
for BChE among slow/fast diastereomers when R is iodide. Increased BChE
inhibitory activity has also been observed for the compound of formula
(I), above, when Rb is a fluoride.

[0098] Accordingly, particularly preferred are the compounds of formula
(Ia), above, wherein the possibilities for X, Y, and Z on the
cycloSal-phospho-triester phenyl ring are as follows: X=H or F, Y=H or
tert-butyl, and Z=H, CH3 or tert-butyl. Preferred also are the
compounds of formula (I) wherein R=CH3 or 125I and
Rb=O-L-DHT, OH, or F. The slow diastereomers also represent the
preferred isomers of the present invention.

[0099] The term "pharmaceutically acceptable salts" as used herein refers
to salts derived from non-toxic, physiologically compatible acids and
bases, which may be either inorganic or organic. Useful salts may be
formed from physiologically compatible organic and inorganic bases,
including, without limitation, alkali and alkaline earth metal salts,
e.g., Na, Li, K, Ca, Mg, as well as ammonium salts, and salts of organic
amines, e.g., ammonium, trimethylammonium, diethylammonium, and
tris-(hydroxymethyl)methylammonium salts. The compounds of the invention
also form salts with organic and inorganic acids, including, without
limitation, acetic, ascorbic, lactic, citric, tartaric, succinic,
fumaric, maleic, malonic, mandelic, malic, phthalic, salicyclic,
hydrochloric, hydrobromic, phosphoric, nitric, sulfuric, methane
sulfonic, naphthalene sulfonic, benzene sulfonic, para-toluene sulfonic
and similar known, physiologically compatible acids. In addition, when a
compound of Formula I contains both a basic moiety and an acidic moiety,
zwitterions ("inner salts") may be formed and are included within the
term "salt(s)" as used herein.

[0100] The compounds of the invention, as stated hereinabove, may be
administered alone, containing both therapeutic and diagnostic moieties,
or alternatively, as two compounds with one compound acting as a
therapeutic and a second compound acting as a diagnostic. The two
compounds could be coadministered concurrently or sequentially. These
compounds may be administered as separate dosage units or formulated for
administration together, according to procedures well known to those
skilled in the art. See, for example, Remington: The Science and Practice
of Pharmacy, 20th ed., A. Genaro et al., Lippencot, Williams &
Wilkins, Baltimore, Md. (2000).

[0101] Therapeutic preparations comprising the compounds of this invention
may be conveniently formulated for administration with a biologically
acceptable vehicle, which may include the patient's own serum or serum
fractions. Other suitable vehicles include liposomes and similar
injectable suspensions, saline, activated carbon absorbents, and
solutions containing cyclodextrins such as alphadex and betadex.
Additionally, IUdR analogs may be derivatized, e.g. by esterification of
available hydroxyl groups, with long chain fatty acids to increase the
circulation half-life of the compounds. The concentration for diagnostic
uses of the compound in the chosen vehicle should normally be from about
0.1 mCi/mL to about 10 mCi/mL. The concentration for therapeutic uses of
the compound in the chosen vehicle should normally be from about 1 mCi/mL
to about 100 mCi/mL. These concentrations may vary depending on whether
the method of administration is intravenous, intraperitoneal, or
intratumoral, which are the preferred routes of administration. In all
cases, any substance used in formulating a therapeutic or diagnostic
preparation in accordance with this invention should be virus-free,
pharmaceutically pure and substantially non-toxic.

[0102] For therapeutic applications, the compound will typically be
administered in a therapeutically effective amount, which will normally
be a dose that provides from about 1 mCi (37 MBq)-20 mCi (740 MBq) of
radioactivity per 24 hours. A diagnostically effective amount of the
compound administered for diagnostic applications will generally be an
amount sufficient to provide between 0.1 mCi and 10 mCi of radioactivity.
For the determination of AR expression, the imaging can commence
immediately after the administration. To detect DNA uptake, imaging may
begin 1 hour after administration. Notably, when using longer lived
radioisotopes, imaging can occur at least daily for 7 days or longer to
assess the tumor growth kinetics. The determination of an appropriate
dose of the compound, either therapeutic or diagnostic, for a particular
patient will, of course, be determined based on the type and stage of the
patient's cancer and the judgment of the attending medical oncologist or
radiologist, as the case may be.

[0103] For both therapeutic and diagnostic applications, the compounds
useful in the method of the invention can be imaged in vitro, ex vivo,
and in vivo by using magnetic resonance spectroscopy or scintigraphic
imaging depending upon the moiety attached to the compound which enables
said imaging. For instance, by labeling a compound of the method of the
invention with .sup.[19]F, the compound could be imaged in vitro, ex
vivo, and in vivo using magnetic resonance spectroscopy (MRS). For
methods of the invention that utilize radiolabeled compounds, those
methods could utilize scintigraphic imaging techniques such as positron
emission tomography (PET) or single photon emission computed tomography
(SPECT).

[0104] As used herein, the expression "tumor activity", refers to a
tumor's presence, progression, regression or metastasis in a subject, or
to a reduction of tumor size due to therapeutic intervention.

[0105] As used herein, the expression, "tumor size", includes all methods
of quantifying the size of a tumor which include, but are not limited to,
weight, mass, and volume of the tumor ex vivo and in vivo. Therefore,
"baseline tumor size", as used herein, refers to the size of the tumor at
or near the time of initial diagnosis, and prior to any form of
treatment, so as to provide a starting point from which changes, or lack
thereof, to the tumor's size can be quantified.

[0106] As used herein, the term "diagnosis" or "diagnostic", includes the
provision of any information concerning the existence, non-existence or
probability of a malignant tumor or a tumor composed of cancer cells in a
patient. It further includes the provision of information concerning the
type or classification of the disorder or of symptoms which are or may be
experience and in connection with it. It encompasses prognosis of the
medical course of the condition.

[0107] If necessary, the action of contaminating microorganisms may be
prevented by various anti-bacterial and/or anti-fungal agents, such as
parabens, chlorbutinol, phenyl, sorbic acid, thimerosal and the like. It
will often be preferable to include in the formulation isotonic agents,
for example, glucose or sodium chloride. Additionally, free-radical
scavengers and antioxidants such as ascorbic acid and the like may be
employed to allow for a longer storage of the radioactive compound.

[0108] The compounds of the invention will typically be administered from
1-4 times a day, so as to deliver the above-mentioned daily dosage.
However, the exact regimen for administration of the compounds and
compositions described herein will necessarily be dependent on the needs
of the individual subject being treated, the type of treatment
administered and the judgment of the attending medical specialist. As
used herein, the terms "patient" and "subject" include both humans and
animals.

[0109] The compounds of the invention may be administered as such, or in a
form from which the active agent can be derived, such as a prodrug. A
prodrug is a derivative of a compound described herein, the pharmacologic
action of which results from the conversion by chemical or metabolic
processes in vivo to the active compound. Prodrugs include, without
limitation, ester, acetal, imine, carbamate, succinate, and phosphate
derivatives of the compounds of formula I, above. Other prodrugs may be
prepared according to procedures well known in the field of medicinal
chemistry and pharmaceutical formulation science. See, e.g., Lombaert et
al., J. Med. Chem., 37: 498-511 (1994); and Vepsalainen, Tet. Letters,
40: 8491-8493 (1999).

[0110] As used herein, the expression "pharmaceutically acceptable carrier
medium" includes any and all solvents, diluents, or other liquid vehicle,
dispersion or suspension aids, surface agent agents, isotonic agents,
thickening or emulsifying agents, preservatives, and the like as suited
for the particular mode of administration desired. Remington: The Science
and Practice of Pharmacy, 20th edition, A. R. Genaro et al., Part 5,
Pharmaceutical Manufacturing, pp. 669-1015 (Lippincott Williams &
Wilkins, Baltimore, Md./Philadelphia, Pa.) (2000)) discloses various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Except insofar as any
conventional pharmaceutical carrier medium is incompatible with the
compounds of the present invention, such as by producing an undesirable
biological effect or otherwise interacting in an deleterious manner with
any other component(s) of a formulation comprising such compounds, its
use is contemplated to be within the scope of this invention. It is noted
in this regard that administration of the compounds of this invention
with any substance that competes therewith for BChE and/or AR binding is
to be avoided.

[0111] The method of treating cancer described herein will normally
include medical follow-up to determine the effectiveness of the
compound(s) of formula (I), above, in eradicating the tumor in a patient
undergoing treatment. The term "treatment", as used herein, refers to
methods of treating malignant tumors or tumors comprising cancer cells
including surgical excision, chemotherapy, and/or radiation therapy.

[0112] Synthetic routes for the preparation of the compounds of formula
(I) are exemplified hereinbelow.

[0113] The targeted delivery of radionuclides to cancer cells in the
manner described herein produces strong cytotoxic activity, in that the
radionuclide is introduced into the DNA of the multiplying cells, where
it induces DNA strand breaks in the double helix. Moreover, by delivering
radiolabeled agents to a specific site and relying on mechanisms
operational at the site of delivery to release the radiolabeled agent,
the usual in vivo degradation pathways are by-passed, bioavailability of
the radiolabeled agent is improved and more tumor cells are exposed to
the cell killing effect of the radiation as they enter into the S phase.

[0114] A kit comprising a vessel containing a compound of formula (I),
above, and a pharmaceutically acceptable carrier medium is also provided.
The kit may optionally include one or more of catheter tubing, syringe,
antibacterial swabs, all antiseptically packaged, as well as instructions
for practicing the above-described methods.

[0115] The following examples describe the synthesis of the compounds of
the present method of the invention, as well as biological testing of
certain of the compounds. These examples are provided for illustrative
purposes only and are not intended to limit the scope of the invention in
any way.

[0117] The cycloSaligenyl monophosphates of
5-[125I]-iodo-2'-deoxyuridine (cycloSal-[125I]IUdRMP) and
5-[125I]iodo-3'-fluoro-2',3'-dideoxyuridine
(cycloSal-[125I]FIUdRMP) were synthesized in consecutive steps, as
shown in Schemes 1 and 2. Nonradioactive iodo-analogs were treated with
hexamethylditin under palladium catalysis to provide the corresponding
5-trimethylstannyl cycloSaligenyl derivatives. These organotin compounds
provided the starting materials for the target [125I]-radioiodinated
cycloSaligenyl phosphotriesters 6b-14b, and 24b. All radioiodolabelings,
proceeding via electrophilic iododestannylation, were carried out at the
non-carrier-added level.

[0118] The cycloSal-moiety was initially inserted into the scaffold as a
chlorophosphite. Thus, 5-iodo-3'-O-levulinyl-2'-deoxyuridine 4, or
optionally IUdR, was treated with cyclic chlorophosphites 15-17 (Scheme
1) in the presence of diisopropylethylamine. The reactions led to
corresponding phosphites which directly oxidized with
tert-butylhydroperoxide and produced the expected diastereomeric mixtures
of cycloSaligenyl products. The same preparation scheme was used to
introduce the cycloSal-moiety into 3'-fluoro-3'-deoxythymidine 20 and
5-iodo-3'-fluoro-2',3'-dideoxyuridine 21, to yield cycloSaligenyl
monophosphates 23 and 24, respectively (Scheme 2). Performing
phosphitylation of IUdR without protecting the 3'- or 5'-OH group on
uridine, simplified the synthesis and allowed for the concurrent access
to the 5'-O- and 3'-O-cycloSal-5-[125I]IUdRMPs, and a parallel
evaluation of biophysical properties in both groups of regioisomers.
Typically, when phosphitylation of IUdR by chlorophosphite (1.05-1.25
molar equivalent) was carried out at a temperature below -40° C.,
a mixture of 5'-O- and 3'-O-cycloSaligenyl phosphotriesters was formed,
with only a marginal regioselectivity. The 3',5'-O,O-diphosphorylation
occurred in the range of 16-22%. The separation of regioisomers, by flash
column chromatography on silica gel, furnished pure diastereomeric 5'-O-,
3'-O- and 3',5'-O,O-cycloSal-products in fair (39-46%) overall yield.
Phosphitylations conducted using IUdR derivatives with protected 3'-OH or
5'-OH groups, did not greatly improve the overall efficiency of the
synthesis due to required protection/deprotection steps. These
independently synthesized regioisomers however, aided the regioisomer's
elution order verification, in purifications of the reaction mixtures
originated from unprotected IUdR. Thus, the 5'-O-phosphitylation of 4,
along with one-pot oxidation, followed by the deprotection of
3'-O-levulinate group, led to the corresponding phosphotriesters 6-8 in
52-62% of the isolated yield.

[0119] The present inventors also examined the introduction of the
cycloSal-component via the direct coupling of cyclic chlorophosphites,
performed at the non-carrier-added concentration level. This approach
permits a straightforward, one-step synthesis of many radiolabeled
cycloSaligenyl monophosphates, starting from derivatives of deoxyuridine
or thymidine already labeled with radionuclide, particularly practical
for [18F]-fluorine, e.g., 3% [18F]-fluoro-3'-deoxythymidine
([18F]FLT).
5-[125I]-Iodo-5'-O-[cyclo-3,5-Di-(tert-butyl)-6-fluoroSaligenyl]-3'--
fluoro-2',3'-dideoxyuridine 24b was prepared through the routine
[125I]-iododestannylation of corresponding stannanes (Scheme 2)
These results confirmed, that the coupling of cyclic chlorophosphites can
be successfully performed at the non-carrier-added concentration level
and may be fully applicable in the preparation of
cycloSaligenyl-[18F]FLT derivatives, or implemented to other
[18F]-radiofluorinated analogs.

[0120] The organotin precursors 6a-14a, and 24a, were acquired using
hexamethylditin (except 6a wherein a tri-n-butyl derivative was used) in
the reactions catalyzed by bis(triphenylphosphine)palladium(II)
dichloride. Stannylations were carried out under nitrogen in boiling
dioxane or ethyl acetate at 60° C., depending on the solubility of
the starting iodotriester. Under these milder conditions, the proton
dehalogenation was reduced from ˜20% to ˜9% at 60° C.
Two major products were consistently obtained. A first product, with a
shorter retention time on TLC and proven to be the trimethylstannyl
derivative, was isolated in 52-77% yield. A second product (with a longer
retention time on TLC) was identified as proton deiodinated starting
phosphotriester. All cycloSaligenyl-5-trimethylstannyl-2'-deoxyuridine
phosphotriesters synthesized in the method of this invention were
amenable to no-carrier-added radio-iododestannylations providing
excellent isolated yields of [125I]-iodolabeled products. Moreover,
a high hydrophobicity of synthesized stannanes in respect to
corresponding iodo-derivatives, allowed for a consistent and complete
separation of the trimethyltin precursor excess from the [125I]-iodo
product, even if a large volume of crude reaction mixture (up to 1 mL)
was loaded onto the HPLC column in final purification.

[0121] All [125I]-radioiodinations were conducted within 0.25-12 mCi
range, using acetonitrile as a solvent and ˜120 μg of the
stannyl precursor. The reaction mixtures were acidified with TFA solution
in acetonitrile and hydrogen peroxide was used to oxidize [125I]
sodium iodide. Modified radio-iododestannylation procedure provided a
consistently excellent radiochemical yield of 85-93% and a radiochemical
purity of ≧95%, across the series of
cycloSaligenyl-phosphotriesters. The proton destannylated product (3-8%)
was found in all of the crude reaction mixtures. The majority of it
originated from frozen tin precursor samples, and increased after the
prolonged storage period of the stannane (sometimes exceeding six
months). In order to confirm the radiochemical purity and precisely
measure the specific activity of products, the reaction mixtures were
purified by HPLC, with parallel monitoring of the radioactivity and
absorbance (220/280 nm). Radiolabeled compounds, if kept in a solution of
aqueous acetonitrile overnight (at concentrations of ˜1
μCi/μL) were routinely re-purified just before conducting intended
experiments. However, the HPLC analysis performed within 24 h generally
indicated the product retained ≧95% of radiochemical purity. The
HPLC co-injections of [125I]-radiolabeled products: 6b-14b, 24b and
corresponding nonradioactive iodo-analogs prepared independently,
positively confirmed the identity of radioiodinated compounds.
Characterization of all non-radioactive phosphotriesters was carried out
by means of 1H, 13C, 119Sn and 31P NMR, as well as
high resolution mass spectroscopy and thorough HPLC analyses.

Experimental Procedures

[0122] Chemicals and reagents were purchased from commercial suppliers and
used without further purification. Anhydrous diethyl ether was distilled
from sodium wire with benzophenone as an indicator and dichloromethane
was distilled from CaH2 under nitrogen. [125I]NaI in
1×10-5 NaOH (pH 8-11) was obtained from PerkinElmer.
Radioactivity was measured with Minaxi γ-counter (Packard, Waltham,
Mass.), a dose calibrator (Cap Intec Inc., Ramsey, N.J.). Analytical TLC
was carried out on precoated plastic plates, normal phase Merck 60
F254 with a 0.2 mm layer of silica, and spots were visualized with
either short wave UV or iodine vapors. Radioactive compounds on TLC and
ITLC plates were analyzed on a Vista-100 plate reader (Radiomatic VISTA
Model 100, Radiomatic Instruments & Chemical Co., Inc., Tampa, Fla.).
Flash column chromatography was carried out using Merck silica gel 60
(40-60 μM) as stationary phase. Compounds were resolved and their
purity confirmed by the HPLC analyses on Gilson (Middleton, Wis.) and
ISCO (Lincoln, Nebr.) systems using 5-μm, 250×4.6 mm, analytical
columns, either Columbus C8 (Phenomenex, Torrance, Calif.) or ACE C18
(Advanced Chromatography Technologies, www.ace-hplc.com). Columns were
protected by guard filters and were eluted at a rate of 0.8 mL/min with
various gradients of CH3CN (10-95%) in water with or without TFA
(0.07%, w/v). Variable wavelength UV detectors UVIS-205 (Linear, Irvine,
Calif.) and UV116 (Gilson) were used with the sodium iodide crystal
Flow-count detector (Bioscan, Washington, D.C.) connected in-line at the
outlet of the UV detector. Both signals were monitored and analyzed
simultaneously. NMR spectra were recorded at ambient temperature in
(CD3)2SO or CDCl3 with a Varian NOVA 500 MHz NMR
instrument spectrometer (Palo Alto, Calif.). Chemical shifts are given as
δ (ppm) relative to TMS as internal standard, with J in hertz.
Deuterium exchange and decoupling experiments were performed in order to
confirm proton assignments. 31P NMR and 119Sn NMR spectra were
recorded with proton decoupling. High resolution (ESI-HR) positive ion
mass spectra were acquired on an LTQ-Orbitrap mass spectrometer with
electrospray ionization (ESI). Samples were dissolved in 70% methanol.
Two μL aliquots were loaded into a 10-μL loop and injected with a 5
μL/min flow of 70% acetonitrile, 0.1% formic acid. FAB high-resolution
(FAB-HR) mass spectra analyses (positive ion mode, 3-nitrobenzyl alcohol
matrix) were performed by the Washington University Mass Spectrometry
Resource (St. Louis, Mo.) and at the University of Nebraska Mass
Spectrometry Center (Lincoln, Nebr.).

General Procedure A (Synthesis of Saligenyl Chlorophosphites)

[0123] To a solution of a dried salicyl alcohol derivative in Et2O
stirred at -16° C. under a nitrogen atmosphere, newly distilled
PCl3 was added. After 15 min, while maintaining the same
temperature, a solution of pyridine in Et2O was added dropwise over
a period of 1 h. The reaction mixture was allowed to reach ambient
temperature and the stirring was continued for further 2 h. To facilitate
a complete separation of pyridinium chloride, the mixture was stored in a
tightly covered reaction flask at -20° C. overnight. After
filtration under pressure of dry nitrogen, a solvent was evaporated in
vacuum and the resulting crude product was used in the next step of
synthesis without a delay.

General Procedure B (Synthesis of Nonradioactive cycloSaligenyl
Phosphotriesters of 5-Iodo-2'-deoxyuridine 6-14, Using Unprotected IUdR
and Chlorophosphites)

[0124] All reactions were performed under anhydrous conditions and a dry
nitrogen or argon atmosphere. To a stirred solution of IUdR and DIPEA
(˜2.5 molar excess) in DMF/THF (2:1 mixture), cooled to or below
-40° C., the THF solution of the appropriate crude chlorophosphite
(1.05-1.25 molar equiv.) was added in small portions. Chlorophosphites
(obtained using General Procedure A) were transferred directly from its
original reaction vessel, by means of an argon pressure and the syringe
equipped with a long double needle. The reaction mixture was then slowly
warmed to ambient temperature and a further stirring continued for 30
min, to ensure a completion of the reaction (TLC monitoring with DCM/MeOH
10:1.0-1.2 range). The reaction mixture was cooled to -40° C. once
again and a solution of tert-butyl hydroperoxide, 5-6 M (2.1-2.5 molar
equiv.) in n-decane was added. The resulting mixture slowly warmed to
room temperature, with the stirring continued for about 1 h (the reaction
progress was followed by TLC). The solvent was evaporated under reduced
pressure and the residue was treated with DCM. The precipitate of
unreacted IUdR was filtered off, a precipitate washed with DCM, dried
under high vacuum. The recovered IUdR was proven suitable for an
immediate reuse. The filtrate was evaporated under reduced pressure and
the residue purified by flash column chromatography on a silica gel,
using a gradient of MeOH in DCM (0.7-1.0:10) and/or a gradient of MeOH in
EtOAc (0.2-0.7:10), to yield each of three formed cycloSaligenyl
regioisomers: 5'-O-, 3'-O- and di-5',3'-O,O-substituted, separated.
Several diastereomers of 5'-O-cycloSaligenyl-, as well as
3'-O-cycloSaligenyl-phosphotriesters were later separated, by means of
the HPLC, giving small quantities (˜30 mg) of each individual
diastereomer.

General Procedure C (Synthesis of Nonradioactive cycloSaligenyl
Phosphotriesters of 5-Iodo-2'-deoxyuridine 6-14, Thymidines and
5-Iodo-3'-dideoxy-3'-fluorouridine, Using Protected IUdR 4 or Uridines,
and Chlorophosphites)

[0125] Under argon or a dry nitrogen atmosphere, DIPEA and the crude
saligenyl chlorophosphite, dissolved in MeCN, were added to a stirred
solution of IUdR 4 in MeCN at -40° C. The reaction mixture was
slowly warmed to ambient temperature and the reaction progress monitored
by the TLC (DCM/MeOH 10:0.7). The reaction mixture cooled one more time
to -40° C. was treated with a solution of tert-butyl hydroperoxide
5-6 M (˜2.5 molar equiv.) in n-decane. The mixture was warmed up
slowly to room temperature and stirred 1-2 h (the reaction progress was
followed by TLC). The solvent was removed under reduced pressure, the
residue treated with DCM (80 mL) or EtOAc (60 mL) and filtered. The
filtrate was washed with the 0.3% aqueous solution of NaHSO3 (20
mL), brine (20 mL), dried over MgSO4 and evaporated. Deprotection
Procedures: in reactions conducted with 4 the resulted solid was
dissolved in pyridine (2 mL) and added to a stirred, cooled on an
ice-water bath, solution of hydrazine hydrate (1.5 mL) in pyridine (3
mL), containing acetic acid (2.2 mL). The stirring continued for 5 min,
and then water (40 mL) and EtOAc (50 mL) were added. The organic layer
was separated and washed with the 10% aqueous solution of NaHCO3 (20
mL), water (20 mL), dried over MgSO4 and evaporated, and the residue
was purified on a silica gel column.

[0126] The solution of appropriate iodouridine 6-14, 21 or 24 (1.0 equiv),
the hexamethylditin (hexa-n-butylditin was used in the preparation of 6a)
(1.25-1.50 equiv) and dichlorobis(triphenylphosphine) palladium II
catalyst (0.10 equiv) in ethyl acetate or dioxane (for 6 and 7) was
refluxed (2-6 h) under a nitrogen atmosphere (until the starting material
disappeared). The reaction progress was monitored by TLC. Two major
products were formed in all the reactions. The first product, with a
higher TLC mobility, isolated in 50-72% yield, was the trialkylstannyl
derivative, and a second one (with a low TLC mobility) was a proton
deiodinated starting compound. After cooling to ambient temperature a
mixture was freed from an excess of the catalyst and partially purified
by the filtration through a thin pad of silica (EtOAc/hexanes, 2:1). The
resulting crude product was purified by repeating a silica gel column
chromatography, using a gradient of EtOAc in hexanes (2-5:10) and/or a
gradient of MeOH in DCM or CHCl3 (0.4-0.7:10). Anhydrous samples of
pure tin precursors (˜100 μg) were stored up to eight months,
with the exclusion of light, under nitrogen at -20° C.; not
showing the excessive decomposition (≦7% by the HPLC analysis) and
were suitable for the immediate radio-iododestannylation. Diastereomers
of 5'-O-cyckSaligenyl-, as well as
3'-O-cyckSaligenyl-5-trimethyltin-phosphotriesters were later separated,
by the HPLC using a reverse phase columns, to give small quantities
(˜20 mg) of each, individual diastereomer.

General Procedure E (Synthesis of [125I]-Radioiodinated
cycloSaligenyl Phosphotriesters of 5-[125I]Iodo-2'-deoxyuridine
6b-14b, and 24b)

[0127] Into a glass tube containing the appropriate tin precursor 6a-14a,
or 24a (100-120 μg, 130-150 μmol), obtained as described
immediately above dissolved in MeCN (50-100 μL), a solution of
Na125I/NaOH (10-100 μL, 1-10 mCi) was added, followed by a 30%
aqueous H2O2 solution (5 μL), and followed by TFA solution
(50 μL, 0.1 N in MeCN) added with a 2 min delay. The mixture was
briefly vortexed and left for 15 min at room temperature. The reaction
was quenched with Na2S2O3 (100 μg in 100 μL of
H2O) and taken up into a syringe. The reaction tube was washed twice
with 50 μL of H2O/MeCN (9:1) solution. The previously withdrawn
reaction mixture, plus washes were injected onto the HPLC system and
separated, by means of C8 or C18 reverse phase column. Eluent from a
column (1 mL fractions collected) was monitored using a radioactivity
detector, connected to the outlet of UV detector (detection at 220 and
280 nm). Eluted fractions containing a product, combined and evaporated
with a stream of dried nitrogen, were reconstituted in an appropriate
solvent and concentration, and were filtered through a sterile (Millipore
0.22 μm) filter into a sterile evacuated vial. Identity of
radiolabeled products was confirmed by the evaluation of UV signals of
nonradioactive iodo-analogs (prepared independently, not through the
iododestannylation reaction) with the radioactive signals, and/or by
comparing Rf obtained from the radio-TLC and tR from the
radio-HPLC analysis. The specific activities were determined by the UV
absorbance of radioactive peaks, as compared to the standard curves of
unlabeled reference compounds. Radiolabeled products, if kept in a
solution overnight at ambient temperature, were repurified before
conducting further experiments, even though HPLC analysis rarely
indicated less than 95% of the radiochemical purity.

[0143] General Procedure E was conducted within 0.54-11.7 mCi range, to
give ˜42 mCi of 6b after six consecutive radioiodinations of the
stannyl precursor 6a. An average isolated yield of the product was 88%.
The latest preparation was carried out with stannane 6a (120 μg) and
[125I]NaI/NaOH (94 μL, 10.2 mCi). The HPLC purification of the
product proceeded on Jupiter C18, 300 {circumflex over (Å)} (5 μm,
4.6×250 mm) column; eluent: solvent A 10% MeCN in water, solvent B
MeCN and 0.8 mL/min elution rate of a linear gradient of B from 0-20%
over 33 min, followed by a linear gradient of B from 20-95% for 5 min,
and finally 95% B for the period of 15 min. The main radioactivity peak
(9.3 mCi, 91% yield) was eluted and collected in four fractions (a total
volume ˜3.3 mL), within 28-32 min after the injection of 460 μL
(˜10.1 mCi) of the reaction mixture. An excess of unreacted tin
precursor 6a was separated from the radioiodinated product without
difficulty, eluting ˜20 min later (tR=50.6 min). If required,
diastereomers of 6b were separated using the same HPLC conditions. The
diastereomer 6b fast eluted at tR=29.8 min and 6b slow at
tR=30.8 min. When a solution of 20% MeCN in water was used as
solvent A and the column was eluted at the 0.8 mL/min flow rate with
solvent A for the period of 25 min, followed by a linear gradient of B
from 0-95% over 10 min, and finally with 95% B for 10 min; diastereomers
were eluted faster and fully separated: 6b fast, tR=20.6 min and 6b
slow, tR=22.4 min. In a single HPLC run, the complete separation
(each diastereomer ≧98% pure, Bioscan NaI(T) detector) was
achieved, if a total amount of injected 6b was ≦230 μCi. Larger
batches of individual diastereomers were acquired by repeating the HPLC
injections, or using a semi preparative column: Columbus C18, 100
{circumflex over (Å)} (5 μm, 10×250 mm), eluted at the 2.6
mL/min flow rate with a 20% MeCN solution in water. Diastereomer 6b fast
eluted within 75-77 min and 6b slow 83-87 min, past the injection, and
each was collected in ˜8 mL volume of an eluent. The solvent was
evaporated to dryness in high vacuum at 30° C., using the SpeedVac
system. The HPLC co-injections of the purified 6b with its nonradioactive
analog 6, and the parallel monitoring of the radioactivity and UV signal,
confirmed the identical elution of both compounds.

[0144] General Procedure E was conducted within 0.52-10.1 mCi range, to
give ˜33 mCi of 7b in six consecutive radioiodinations of the
stannyl precursor 7a. An average isolated yield was 81%. The latest
radioiodination was performed with stannane 7a (˜110 μg) and
[125I]NaI/NaOH (90 μL, 9.3 mCi). The HPLC purification of the
product continued on Jupiter C18, 300 {circumflex over (Å)} (5 μm,
4.6×250 mm) column; eluent: solvent A 18% MeCN in water, solvent B
MeCN; and 0.8 mL/min flow rate. The column was eluted with solvent A for
the period of 30 min, then with a linear gradient of B from 0-95% over 10
min, and 95% B for min. The product 7b (8.1 mCi, 87%), which eluted
within 21.5-24.7 min after the injection of 410 μL (˜9.1 mCi) of
the reaction mixture, was collected in three fractions (2.5 mL a total
volume). An excess of unreacted tin precursor 7a was fully separated,
eluting between 27.3-27.7 min. The HPLC co-injections of the purified 7b
with its nonradioactive analog 7, and monitoring the radioactivity
(Bioscan NaI(T) detector) and UV signal at 280 nm, verified the same HPLC
mobility of both compounds. Diastereomers of 7b were separated on Jupiter
C18, 300 {circumflex over (Å)} (5 μm, 4.6×250 mm) column,
eluted with 20% MeCN solution in water, for the period of 45 min at the
0.8 mL/min flow rate. Individual diastereomers: 7b fast (tR=25.3
min) and 7b slow (tR=26.8 min) were ≧98% pure (Bioscan
NaI(T)). In the course of a single HPLC run, the full separation of
diastereomers was limited to the total of ˜270 μCi 7b loaded
onto a column. Larger batches of resolved diastereomers were attainable
through the repetitive HPLC injections, or using a larger column:
Columbus C18, 100 {circumflex over (Å)} (5 μm, 10×250 mm);
eluent: a solution of 22% MeCN in water and the flow rate of 2.5 mL/min.
Diastereomer 7b fast eluted within 72-77 min, and 7b slow within 79-81
min past the injection. The solvent was evaporated to dryness in high
vacuum, on the SpeedVac system. The product residue was analyzed again on
the analytical HPLC shortly before conducting scheduled experiments and
was reconstituted in an appropriate solvent.

[0145] The overall ˜52 mCi of 8b was acquired in eleven successive
radioiodinations, using one of the purified tin precursors: 8a, 8a fast
or 8a slow, and conducting General Procedure E within the 0.25-10.7 mCi
range. An average isolated yield of the product was 88%. The latest
radiolabeling was performed with diastereomeric stannane 8a (˜115
μg) and [125I]NaI/NaOH (40 μL, 3.91 mCi). The HPLC
purification of the product proceeded on ACE C18, 100 {circumflex over
(Å)} (5 μm, 4.6×250 mm) column; eluent: solvent A 50% MeCN
in water, solvent B MeCN; and the flow rate of 1.0 mL/min. A column was
eluted with solvent A for the period of 60 min, then with a linear
gradient of B from 0-95% over 10 min, and 95% B for 20 min. The product
8b (3.48 mCi, 89%), which eluted within 25-29 min after the injection of
350 μL (˜3.76 mCi) of the reaction mixture, was collected in
four fractions. An excess of unreacted stannane 8a was eluting ˜15
min later. The mixture of purified 8b (˜12 μCi, 10 μL) and
the corresponding nonradioactive analog 8 (˜15 μg, 20 μL) was
prepared in acetonitrile, and injected onto the HPLC, using the same
column and conditions as during the separation of the product. Both
compounds eluted together, showing the identical retention times.
Diastereomers of 8b were separated on ACE C18, 100 {circumflex over
(Å)} (5 μm, 4.6×250 mm) column, eluted at the 0.8 mL/min
flow rate, with a 45% MeCN solution in water, for the period of 45 min.
Each diastereomer: 8b fast (tR=30.3 min) and 8b slow (tR=32.8
min), was ≧98% pure (Bioscan NaI(T)). The complete separation of
diastereomers in the single HPLC run was reached, if the total amount of
8b loaded onto the column was ≦220 μCi.

[0146] Larger batches of the individual diastereomers were obtained by the
repetitive HPLC injections of purified 8b, or by conducting the
radioiodination using a single diastereomer of the tin precursor 8a fast
or 8a slow. Fractions containing the product were combined and the
solvent was evaporated in high vacuum on the SpeedVac system. The product
residue was reconstituted in MeCN and analyzed again on the analytical
HPLC, shortly before conducting planned experiments.

[0147] The overall amount of prepared 24b was 10.4 mCi, acquired in four
consecutive radioiodinations. General Procedure E was carried out within
the 0.5-5.2 mCi range and an average isolated yield was 93%. The largest
conducted radiolabeling proceeded with stannane 24a (˜120 μg)
and [125I]NaI/NaOH (60 μL, 5.2 mCi). The HPLC purification of the
crude product was best achieved on ACE C18, 100 {circumflex over (Å)}
(5 μm, 4.6×250 mm) column; eluent: solvent A 50% MeCN in water,
solvent B MeCN. A column was eluted at 1.0 mL/min of the flow rate, with
a linear gradient of B from 0-95% over 60 min, followed by 95% B for the
period of 30 min. The product 24b (4.85 mCi, 92%) collected within 26-28
min after the injection of 500 μL (˜5.1 mCi) of the reaction
mixture, was fully separated from an excess of the tin precursor 24a,
which eluted ˜9 min later (37.0-37.6 min). Fractions containing the
product were combined, solvent evaporated with a stream of nitrogen, and
the residue further dried in high vacuum. The mixture of purified 24b
(˜12 Ci, 10 μL) and its nonradioactive analog 24 (˜15
μg, 20 μL) was prepared in acetonitrile and analyzed on the HPLC,
using the same setting as during the separation of the product. The
analysis showed diastereomer 24b fast at tR=26.1 min and 24b slow;
tR=26.7 min, co-eluting with the [127I]-iodoanalog: 24 fast,
tR=25.9 min and 24 slow, tR=26.5 min. Diastereomers of 24b were
separated on ACE C18, 100 {circumflex over (Å)} (5 μm,
4.6×250 mm) column; eluent: solvent A 50% MeCN in water, solvent B
MeCN. A column was eluted with a linear gradient of B from 0-10% over 60
min at the 0.8 mL/min flow rate. Each diastereomer, 24b fast
(tR=38.1 min) and 24b slow (tR=40.7 min), was ≧98% pure
(Bioscan NaI(T) detector). In the single HPLC run a full separation was
possible, if the total amount of 24b loaded onto a column was ≦120
μCi. Larger lots of single diastereomers were obtained by the
repetitive HPLC injections or using a larger column: Columbus C18, 100
{circumflex over (Å)} (5 μm, 10×250 mm); eluted at the 2.2
mL/min flow rate with 57% MeCN in water.

[0148] Since the synthesis of cycloSaligenyl-phosphotriesters is not
stereoselective, all products are obtained as mixtures of two
diastereomers (SP and RP configuration, approximately 1:1
ratio) at each phosphorus center. The diastereomers are differentiated
and named as the -fast and the -slow diastereomer, in correlation to the
HPLC retention time (tR) of each isomer. Only a tentative assignment
of the RP/SP stereochemistry at the phosphorus can be made, by
the association to previously synthesized cycloSal-compounds, with
already known stereochemical configuration (Balzarini J, Aquaro S,
Knispel T, Rampazzo C, Bianchi V, Perno C F, De Clercq E, Meier C.
Cyclosaligenyl-2',3'-didehydro-2',3'-dideoxythymidine monophosphate:
efficient intracellular delivery of d4TMP. Mol. Pharmacol. 2000 November;
58(5):928-35), as a reference. Diastereomers of all synthesized
5'-O-cycloSaligenyl-phosphotriesters could be separated by the reverse
phase HPLC. The compounds of the present invention, 6b-14b, 23, and 24b
were analyzed by reverse phase HPLC to determine their diastereomeric
purity (FIGS. 1-5).

[0149] The individual [125I]-radioiodinated diastereomers of
5'-O-cycloSal-triesters 6b-8b and 3'-O-cycloSal-triesters 12b-14b were
prepared in one of two accessible ways: 1) conducting
[125I]-iododestannylation with a single diastereomer of
trimethylstannyl-precursors 6a slow-8a slow or 6a fast-8a fast, or by 2)
using a diastereomeric mixture of stannane and the separation of isomers
during a final purification of the [125I]-radioiodolabeled product.
The complete separation of [125I]-labeled diastereomers was limited
however, to a total of ˜560 μCi injected in a single HPLC run.
Therefore, if a larger batch of a single [125I]-iodolabeled
diastereomer was required; the individual diastereomer of trimethyltin
precursor was preferred, to speed up the preparation. Diastereomers of
125I-radioiodolabeled cycloSaligenyl-triesters were regularly
obtained in quantities of up to 10 mCi, starting with the selected
diastereomer of appropriate tin precursor.

[0150] All 3',5'-O,O-di-cycloSaligenyl-phosphotriesters 9-11 products,
with two different stereogenic centers formed during the synthesis, were
expected to exist as mixtures of four stereoisomers (with configurations:
SP/SP, RP/SP, SP/RP and RP/RP)
all in a ratio of 1:1. Indeed, the HPLC analysis and 31P NMR
spectroscopy of 3',5'-di-substituted iodides 9-11 and stannanes 9a-11a
pointed out to mixtures of four diastereomers. These diastereomers were
practically inseparable and all of the applied HPLC methods led only to a
partial separation of the slowest or the fastest isomer. Consequently,
the 125I-labeled target 3',5'-O, O-dicycloSaligenyl-phosphotriesters
9b-11b were purified and isolated as mixtures of diastereomers.

Example 3

Synthesis of a O-succinyl-dihydrotestosterone analog of a corresponding
5'-O-cyclosaligenyl-2'-deoxyuridine monophosphate

##STR00010##

[0152] Nonradioactive analogues, containing the androstan-3-one moiety
were all prepared by esterification of dihydrotestosterone
17β-succinate with the corresponding
5'-O-cyclosaligenyl-2'-deoxyuridine monophosphates (Scheme 3). Synthesis
of radiolabelled analogues was based on the non-carrier-added
electrophilic destannylation of the related trialkyl-organotin
precursors, which were prepared by the stannylation of iodouridines,
using hexamethylditin, and were carried out in the presence of
palladium(II) catalyst.

[0155] Into a glass tube containing
5-trimethylstannyl-5'-O-cyclo(3-methylsaligenyl)-3'-O-(17β-succinyl--
5α-androstan-3-one)-2'-deoxyuridine monophosphate (120 μg,
˜150 μmol) dissolved in MeCN (50 μL), a solution of
Na125I/NaOH (10 μL, 0.6-2.0 mCi) was added, followed by a 30%
water solution of H2O2 (5 μL), and by TFA solution (50
μL, 0.1 N in MeCN) added with a 2 min delay. The mixture was briefly
vortexed and left for 15 min at room temperature. The reaction was
quenched with Na2S2O3 (100 μg in 100 μL of H2O)
and taken up into a syringe. The reaction tube was washed twice with 50
μL of H2O/MeCN (9:1) solution. The previously withdrawn reaction
mixture, plus washes were injected onto the HPLC system and separated, by
means of Jupiter C18, 300 {circumflex over (Å)} (5 μm,
4.6×250 mm) column; eluent: solvent A 50% MeCN in water, solvent B
MeCN; and a column eluted at 0.8 mL/min with a linear gradient of B from
0-50% over 45 min. The eluent from a column (1 mL fractions collected)
was monitored using a radioactivity detector, connected to the outlet of
UV detector (detection at 220 and 280 nm). The reaction was conducted
four times within 0.64-1.87 mCi range and an average isolated yield of
the product was 88%. The main radioactivity peak (80-91%) was eluted and
collected in four fractions (a total volume ˜3.3 mL), within
29.5-33 min after the injection of 460-500 μL of the reaction mixture.
An excess of unreacted tin precursor was separated from the
radioiodinated product without difficulty, eluting ˜12 min later.
Eluted fractions containing a product, combined and evaporated with a
stream of dried nitrogen, were reconstituted in MeCN (˜1 μCi/1
μL concentration) and were filtered through a sterile (Millipore 0.22
μm) filter into a sterile evacuated vial. Identity of radiolabeled
product was confirmed by evaluating the UV signal of nonradioactive
iodo-analog with the radioactivity signal of the product, from the
radio-HPLC analysis. The specific activities were determined by the UV
absorbance of radioactive peaks, as compared to the standard curves of
unlabeled reference compound.

[0156] Assays employed to determine IC50 were developed by the
present inventors and utilized UV based detection. For UV-based assays, a
BChE solution in 0.1 M potassium phosphate, pH 7.0 was placed in the
desired numbers of wells of a 96-well plate (0.05 mL/well). The
investigated compound was diluted in DMSO to produce concentrations from
0 to 10 μM in DMSO and 0.002 mL/well of these dilutions was added to
BChE-containing wells. Reaction mixtures were incubated at room
temperature for 30 min. The reagent consisting of BChE substrate, 1 mM
(2-mercaptoethyl)trimethylammonium iodide butyrate, and 0.5 mM
5,5'-dithio-bis(2-nitrobenzoic acid) in 0.1 M potassium phosphate, pH 7.0
(prepared fresh for each assay) was added, 0.25 mL per well. The mixture
was incubated at room temperature and the OD at λ=405 nm was read
at 1, 2, 3, 4, 5, 10, and 15 min after the addition of this reagent using
the Opsys MR®plate reader (Dynex Technologies, Chantilly, Va.). Data
were analyzed using the IC50 nonlinear regression function provided
by GraphPad Prism (GraphPad Software, La Jolla, Calif.).

[0157] The compound binding of several non-radioactive compounds of the
method of the invention were analyzed by the aforementioned human BChE
compound binding study to determine their affinity for the therapeutic
target (Table 1).

[0158] The binding study demonstrates a definite preferential trend for
the slow isomer rather than the fast isomer of the given compounds of the
invention.

Example 5

Biodistribution and Pharmacokinetics of Representative Radioactive
Compounds of Formula (I)

[0159] The in vivo properties and pharmacology of several compounds of the
present invention were initially tested in athymic mice bearing
subcutaneous LS174T tumors (human colorectal adenocarcinoma), which have
several desirable properties such as rapid growth when implanted
subcutaneously (SQ), good vascularization and the availability of
considerable historical data for the parent compound IUdR. The clearance
rates from blood, tumor, and associated organs were analyzed to determine
the structure-pharmacokinetics relationship. The first compounds studied
were 6b-fast and 6b-slow. Four-to-six-weeks old female athymic NCr-nu/nu
mice (NCI-Frederic, Md., USA) were allowed to acclimate in Applicants'
facility after delivery for no less than 5 days. All protocols involving
animals were approved by the University of Nebraska Institutional Animal
Care and Use Committee. Mice were housed in microisolator cages with free
access to sterilized standard rodent diet and water. LS174T cells in 0.1
mL of serum-free medium were implanted SQ at 5×106
cells/mouse. One week later identification transponders were implanted,
also SQ. Body weights and tumor sizes were monitored twice weekly, and
approximately 10 days after the cell implant, mice were randomized for
biodistribution studies.

[0160] Each of the two isomers, 6b-fast and 6b-slow, was tested
independently in separate groups of mice. The comparison to the parent
compound, IUdR, was made by co-injecting 131IUdR with a particular
125I-labeled diastereomer. Compounds were administered intravenously
(IV) via a tail vein.

[0161] Biodistribution was conducted at 1 h, 4 h, 24 and 48 h after the
administration of radiolabeled compounds (n=6 per time point). The
injection doses contained approximately 1 μCi (37 kBq) 131IUdR
and 5 μCi (185 kBq) 125I-labeled 6b-fast or 6b-slow dissolved in
0.2 mL phosphate buffered saline containing 0.1% bovine serum albumin, pH
7.2 (PBS). All syringes were weighted after loading with the compound
solution and immediately after the injection to determine the weight of
the injected dose. Triplicate standards of the injected dose were
prepared in PBS and counted in a gamma counter just before the beginning
of the experiment. The radioactive content of these standards was also
determined alongside all tissues after the necropsy to correct the tissue
uptake for the decay of the radioisotope. Blood, liver, spleen, heart,
lungs, kidneys, brain, tumor and tail were collected during necropsy.
Tissues were rinsed in ice-cold saline, patted dry and weighed. The tail
was collected to validate the quality of the IV injections.

[0162] Clearance curves of 6b slow (FIG. 6A) and 6b fast (FIG. 6B) in
tumor, harvested from athymic mice bearing SQ LS174T xenografts were
prepared. The most significant finding shown in FIG. 6 is that the tumor
uptake and retention of 6b is nearly 14× and 7× higher
compared to the tumor uptake of the parent drug 131IUdR at 24 hours
and 48 hours after administration, respectively. Liver and lungs also
retain high levels of radioactivity. The clearance from these tissues
appears to parallel the levels of 6b in blood.

[0163] Biodistribution studies of 6b slow, 7b fast, 7b slow, 8b fast, and
8b slow have also been completed. The in vivo fate 7b fast and 7b slow is
very similar to the behavior of 6b fast and 6b slow and the observed
differences follow the hydrophobicity of these two derivatives.

[0164] FIG. 7 presents whole body images for a typical time course
distribution (24 h, 48 h, and 72 h) of 6b administered as a
diastereomeric mixture in LS174T-bearing athymic mice. The images were
acquired 1, 2, and 3 days after the administration of the compound. It is
apparent that the radioactivity in tumor and several normal organs
persists, however, it is also apparent that normal organs such as liver
clear the compound at a much faster rate than the tumor, which retains
the compound. It is also apparent that based on pharmacokinetics, the use
of these compounds can be tailored to a specific malignancy and its
anatomical location.

[0165] Images shown in FIG. 8 illustrate the fate of 8a fast in LS174T
tumor model indicating that the compounds with lower BChE activity are
useful in rapidly proliferating tumors. Compounds 8b fast and 8b slow
have a distinct distribution pattern, unlike any of the other isomers.
The distinct behavior of these compounds in the tumor, blood, and several
normal tissues is illustrated in FIGS. 9 and 10 for compounds 8b fast and
8b slow, respectively. Based on the biodistribution and imaging studies,
it is apparent that depending on the application, the compounds of the
invention have "ideal" in vivo properties, i.e., 8b slow has a rapid
clearance from blood and normal tissues and therefore it is suitable for
the imaging of tumor response to therapy or for cancer diagnosis via a
systemic administration. Other compounds have prolonged presence and
therefore are more suited for loco-regional administration and are ideal
for therapy.

[0166] Four compounds, 6b fast, 6b slow, 7b fast, and 7b slow, were
initially selected for a pilot therapy protocol in the intraperitoneal
model of ovarian adenocarcinoma using OVCAR-3-bearing athymic mice. Based
on similar biodistribution of 6b fast, 6b slow, and 7b fast, 7b slow,
pairs, the therapy was conducted using the diastereomeric mixtures. 8b
fast and 8b slow are evaluated individually in therapy trials. It was
particularly important to have these two isomers separated because their
in vivo behavior is radically different, including their blood clearance,
and liver and lung uptake levels after the IV administration.

[0167] In pilot therapy experiments, the effects of unmodified
125IUdR on the growth of IP OVCAR-3 tumors were compared to the
therapeutic effects of the 6b diastereomeric mixture and 7b
diastereomeric mixture. The tumor model used in this study, OVCAR-3 cell
line, was established from the malignant ascites of a patient with
progressive adenocarcinoma of the ovary after many types of chemotherapy
including cyclophosphamide, adriamycin, and cisplatin. OVCAR-3 is
resistant in vitro to clinically relevant concentrations of adriamycin,
melphalan, and cisplatin (Hamilton T C, Young R C, Louie K G, Behrens B
C, McKoy W M, Grotzinger K R, Ozols R F. Characterization of a xenograft
model of human ovarian carcinoma which produces ascites and
intraabdominal carcinomatosis in mice. Cancer Res. 1984; 44:5286-90.). In
immunodeficient mice, IP injected OVCAR-3 cells produce malignant
ascites, peritoneal carcinomatosis, and serosal and visceral seeding
that, if left untreated, leads to death from respiratory compromise,
hemorrhage from invasion of intra-abdominal blood vessels, and bowel
obstruction. Substantial literature data (nearly 100 publications)
confirm that this tumor model has pathogenesis and metastatic properties
similar to those of human ovarian cancer.

[0168] Mice received IP implants of approximately 2.4×108
OVCAR-3 cells/mouse isolated from fresh OVCAR-3 mouse ascites. Cell
viability was measured before and after injection, and was ≧90%.
Four days after tumor cells were implanted, the mice received SQ
transponders and were randomized via a lottery into four groups:
NT--untreated controls that received IP injection of PBS; 7b
group--receiving IP doses of the 7b diastereomeric mixture in PBS
(average 0.4 mCi/mouse (14.8 MBq)); 6b group--receiving IP doses of the
6b diastereomeric mixture in PBS (average dose 0.36 mCi/mouse (13.3
MBq)); and 125IUdR group--receiving IP doses of 125IUdR
(average 0.43 mCi/mouse (15.9 MBq)) in PBS. Mice were monitored three
times per week.

[0169] When the body weight and the gross observation of the mice,
including the palpitation of the abdomen, indicated that some solid
tumors are beginning to form, mice were given a boost dose of the
compounds as follows: 26 days after the first dose mice in the 7b group
were treated with additional 0.18 mCi/mouse (6.6 MBq) 7b diastereomeric
mixture in PBS. Mice in the 6b group received 0.22 mCi (8.1 MBq) 6b
diastereomeric mixture on day 27; and on day 28 mice in 125IUdR
group were treated with additional 0.2 mCi (7.4 MBq) 125IUdR. Slight
differences in the dosing schedule and the administered doses are because
of the required large quantities of the compounds. For these experiments,
˜20 mCi of no-carrier added and radiochemically pure compounds were
required. Each agent was purified on the analytical reversed phase HPLC
column. The use of preparative columns in this case is impractical
because the products are recovered in large volumes of
acetonitrile-containing solvent that must be evaporated to dryness before
the use in mice. The resolution on the analytical column is superior and
the collected volumes much smaller.

[0170] Overall, mice in the 7b group were treated with an average of 0.58
mCi (21.5 MBq) 7b diastereomeric mixture; mice in the 6b group were
treated with an average of 0.58 mCi (21.5 MBq) 6b diastereomeric mixture,
and mice in the 125IUdR group were treated with an average of 0.63
mCi (23.3 MBq) 125IUdR.

[0171] The therapy was terminated 7 weeks after the first dosing. Mice
were sacrificed for the biodistribution study. Blood and solid tumor were
taken for the evaluation of their radioactive content. The peritoneal
cavity was lavaged with 2-mL aliquots of PBS, and PBS wash and ascites
were collected. One mL of the ascites suspension with cancer cells was
taken for gamma counting. The rest of the abdominal fluid was centrifuged
and 0.5 mL of the supernatant was reserved for gamma counting. The weight
of the cell pellet and the supernatant were determined.

[0172] FIG. 11A summarizes the weights of solid tumors as the tumor burden
in addition to the cell pellets recovered from the abdominal cavities of
these mice. There is a significant reduction, ˜50%, in solid tumor
deposits in the 6b-treated and 7b-treated mice as compared to
125IUdR. The statistical analyses of these data are shown in Table
2.

[0173] Two weeks after the first dose of radiolabeled compounds, the whole
body radioactivity was measured to determine to what extent the
additional doses of the compounds may increase the radiation burden and
therefore may be detrimental to the animal health (FIG. 11B). The
absolute amounts were low and ranged from -6.5 μCi for the 6b group to
-4 μCi in the 7b group. It also appears that notwithstanding great
similarities in the in vivo distribution of 6b fast, 6b slow and 7b fast,
7b slow, the whole body retention after 14 days is distinctly higher for
the 6b group, with the following specific values: 1.87 (0.16) % ID
(median 1.89% ID) for 6b; 1.21 (0.12) % ID (median 1.25% ID) for 7b; and
0.97 (0.11) % ID (median 1.08% ID) for 125IUdR. The corresponding P
values are 0.003, 0.0001, 0.166 for 7b vs. 6b, 6b vs. 125IUdR, and
7b vs. 125IUdR, respectively.

[0174] The data collected after necropsy conducted 4 weeks after the boost
dose indicated that the tumor retention of the 6b and 7b was
significantly higher compared to 125IUdR (FIG. 12). Percent injected
doses (ID) shown in FIG. 12 were calculated based on the cumulative
administered dose and are uncorrected for decay.

[0175] Biodistribution data for 8b showed a very favorable blood clearance
curve (FIGS. 9 and 10). It also indicated that the liver is the major
site of the normal tissue uptake. Therefore, it was expected that 8b slow
might prove to be an excellent choice for the IP therapy. Having already
established that four compounds are effective in the early stages of the
OVCAR-3 tumor development on athymic mice, this therapy was designed to
commence at the advanced stages of the disease.

[0176] Female athymic NCr-nu/nu mice were received from NCI-Frederic (MD,
USA). Identification transponders were implanted SQ in mice acclimated
for one week. When mice reached the age of 8-weeks, all mice received IP
implant of 5.6×108 OVCAR-3 cells/mouse in 0.5 mL media without
serum. This cancer cell load is double the size of implant described in
Example 6 and allows for a rapid development of the advanced stages of
the tumor. One weeks later mice were separated into three groups via a
lottery as follows: mice in the control group were left untreated; mice
the vehicle group received 0.3 mL vehicle (PBS containing 0.5% albumin
and 5% DMSO); the remaining mice in groups ×1, ×2, and
×3 received an IP injection of 8b-slow in 0.3 mL vehicle; two weeks
later mice in groups ×2 and ×3 received an IP injection of
8b-slow in 0.3 mL vehicle; and four weeks after the first dose mice in
group ×3 received an IP injection of 8b-slow in 0.3 mL vehicle.
Using the same schedule, mice in the vehicle group were given IP
injections of 0.3 mL vehicle. The average total administered doses were
as follows: group ×1: 0.5 mCi (17.5 MBq); group ×2: 1 mCi (37
MBq), and group ×3: 1.5 mCi (55.5 MBq). All mice were killed six
weeks after the administration of the first dose. FIG. 13 shows the
weights of tumors extirpated from these mice and collected in the
peritoneal lavage. FIG. 14 shows a unambiguous dose-response relationship
between the tumor size and the total administered doses of 8b slow. The
hemoglobin and hematocrit data shown in FIG. 15 indicates that these
doses of radioactivity do not produce any significant hematological
adverse effects. The data analyses confirmed that the observed
differences are statistically significant. The table shown in FIG. 16
summarizes P values for all treatment groups.

Example 8

Dose-Response Therapy in Early State OVCAR-3 Tumors

[0177] Female athymic NCr-nu/nu mice were received from NCI-Frederick (MD,
USA) and were allowed to acclimate in Applicants' facilities until the
age of 8 weeks. After this period of acclimation, 2.5×108
OVCAR-3 cells were implanted into 36 mice. Four days later, each mouse
received SQ transponders (micro-identification chips) and mice were
divided into three groups at random: control mice receiving sham IP
injections of 0.6 mL vehicle (n=12); mice in the high dose group were
treated with one IP dose of 0.7 mCi (26 MBq) 8b slow in 0.6 mL vehicle
(n=12); and mice the low dose group were treated with one IP dose of 0.29
mCi (10.7 MBq) 8b slow in 0.6 mL vehicle (n=12).

[0178] Five weeks after treatment, all mice were sacrificed and full
necropsy was performed. A single dose of 8b slow given at the early
stages of the disease reduces the tumor burden by >230%. The response
of solid tumors seems better than the cells in ascites, >300%
reduction in the weight of the solid tumor deposits (FIG. 17). The uptake
and retention of radioactivity was measured in several tissues and in
tumor. The radioactivity levels in blood are ˜30 times lower than
in the tumor cells in ascites and ˜10 times lower than in the solid
tumors. The hemoglobin and hematocrit levels indicated that the doses
used in this therapy were not causing noticeable adverse effects. It is
also encouraging that the liver uptake was low, <0.003% ID/g,
indicating that the IP administration bypasses some of the hepatic
metabolism of this compound.

[0179] For each compound tested, LS174T cancer cells were plated in four
6-well plates at 2×105 cells/well in 3 mL growth medium. After
24 h in culture, radioactive compounds were added to wells at
predetermined times. The cells were incubated with compounds up to 360
min. Each point in time was tested in triplicate. Aliquots of media (0.5
mL) were removed from each well for gamma counting to determine the
radioactive concentration in each well. At the end of incubation, the
radioactive medium was aspirated and disposed. Cells were washed twice
with 3 mL ice-cold PBS. Aliquots of wash PBS (0.5 mL) were also taken for
gamma counting. Cells were trypsinized with 1.5 mL trypsin/EDTA and 1-mL
aliquot of the cell suspension was counted in a gamma counter. FIGS. 18
and 19 demonstrate the uptake kinetics of 7b fast and 7b slow in OVCAR-3
ovarian cancer cells and LS174T colorectal cancer cells, respectively.
The data indicates that the uptake is governed by the levels of BChE
expression.

[0180] LS174T cells, 2×106 cells/flask, were plated in T-75
flasks. After ˜18 hours in culture, the growth medium was removed
from all flasks and replaced with either 15 mL fresh medium containing 6b
fast (114.5±0.17 kBq/mL; 3.09±0.004 μCi/mL) or 15 mL fresh
medium containing 6b slow (114.4±0.35 kBq/mL; 3.09±0.010
μCi/mL). Control cells were given 15 mL fresh nonradioactive medium.
Triplicate 0.1-mL aliquots of medium were withdrawn from each flask and
counted in a gamma counter to determine the concentration of radiolabeled
compounds. After 4 hours in the incubator at 37° C., the growth
medium was removed from all flasks. Monolayers were washed once with
medium and 15 mL fresh medium was added to each flask. Cells were allowed
to grow undisturbed for 24 hours at which time cells were trypsinized,
counted, and their viability was determined. Cells were re-plated in T-25
flasks at plating densities of 500 cells/flask and 200 cells/flask. Each
cell density was tested in quadruplicate. Seventeen to 21 days later,
colonies were washed with 5 mL ice-cold PBS, followed by 5 mL
PBS/methanol (1:1; v/v), and fixed in 5 mL methanol for 10 min. Methanol
was discarded and flasks were left open to dry for a few hours. Crystal
violet (5 mL; 0.25% in 1:1 PBS/methanol) was added to each flask to stain
cells. After approximately 10 min, the dye was removed and flasks were
rinsed first with tap water followed by distilled water, and were left to
dry. Colonies were counted manually by two observers using the Wheaton
colony counter (Wheaton, Millville, N.J., USA). FIG. 20 shows the
surviving fractions calculated as the ratio of the number of colonies
derived from cells treated with the radioactive compounds to the number
of colonies derived from untreated control cells.

[0181] An alternative procedure with a higher concentration of the
radioactive compounds and a shorter exposure time to the radioactive
compounds was also employed. LS174T cells (2×106) were plated
in T75 flasks and allowed to grow for ˜18 hours, at which time the
medium was removed and replaced with the medium containing radioactive
compounds 7 fast and 7 slow at 5 μCi/mL concentration. Cells in
control flasks received non-radioactive media. Triplicate 0.1-mL aliquots
were taken from each flask for gamma counting. Cells were returned to the
incubator for 4 hours. The medium was removed from all flasks, including
controls, and the cell monolayer was washed once with fresh
non-radioactive medium without FBS. Five mL 2.5% trypsin-EDTA was added
each flask to dissociate the monolayer. Fresh FBS-containing medium was
added to stop the action of trypsin and to form a single cell suspension.
Cell numbers and cell viability were determined. All cell suspensions
were centrifuged at 800 rpm for 10 min at 4° C. FBS-containing
medium was added to cell pellets to produce 1×106 cells/mL
suspension. One mL of each suspension was counted in a gamma counter to
determine cell-associated radioactivity. Cells were plated in duplicate
T25 flasks at densities of 1,000 cells/flask, 500 cells/flask, and 100
cells/flask. The media was changed approximately once a week. After three
weeks, colonies were processed as described above (FIG. 21). The
cytotoxicity of these drugs appears to be BChE-dependent and it parallels
the DNA uptake and retention.

[0182] OVCAR-3 cells were plated into six flasks and allowed to attach
overnight. The growth medium was removed and replaced with 10 mL fresh
medium containing radioactive compounds. Cells were exposed to the
compound for 1 hour after which time the radioactive medium was removed
and replaced with 12 mL fresh medium. Aliquots of all radioactive growth
media were counted in the gamma counter. Cells in three flasks were
processed immediately. The cells in the remaining three flasks were
cultured for 24 hours and then processed. The cell monolayer was rinsed
with 5 mL PBS; trypsinized, cell were counted and their viability
determined. Using NE-PER nuclear and cytoplasmic extraction reagents,
cell content was fractionated and counted in a gamma counter to determine
the compound distribution in various compartments of the cancer cell.
FIG. 22 shows the subcellular distribution of 6b in OVCAR-3 cancer cells.
This example illustrates how the selection of either fast or slow
radiolabeled compounds can be tailored to the specific rate of cancer
cell proliferation.

Example 12

Cell Survival of U-87 Human Glioblastoma after Treatment with 6b

[0183] U-87 MG cell line is an epithelial cell line derived from a grade
IV glioblastoma resected from the brain of a 44 years old, female
Caucasian patient. U-87 cells were plated in four flasks for each isomer
and allowed to attach for 24 h. The compounds 6b fast and 6b slow were
added to U-87 cells at 1 Ci/mL (37 kBq/mL). Cells were incubated with the
radioactive compounds for 24 h and the radioactive medium was removed.
Cells were washed twice with fresh, nonradioactive medium and
trypsinized. After appropriate dilutions in full growth medium, cells
were re-plated in fresh medium (n=4 per compound). After 96 hours in
culture, cells were harvested and their numbers counted using a
Cellometer® disposable cell counting chamber (Nexcelom Bioscience,
Lawrence, Mass.). Four flasks used as controls were sham-treated with a
volume of PBS used to dissolve radioactive compounds. The control flasks
were processed as described for the radioactive compound-treated cells.
FIG. 23 demonstrates the surviving fractions of cells treated with both
isomers of 6b. The surviving fraction is calculated as the ratio of cell
number recovered from flasks treated with radioactive compounds to the
cell number recovered from the control flasks. Averages and standard
deviations are shown. The cell survival was also measured in a 96-well
and 6-well formats.

[0184] U-87 cells were plated in 6-well plates from suspension. The
compounds 6b fast and 6b slow were added with media on day 0. Twenty-four
hours later, radioactive medium was removed and fresh, full growth medium
was added. Cells were grown for 72 h, medium was replaced and cells
continued to grow for additional 72 h. Growth medium was removed; cell
monolayers were washed with ice-cold PBS, followed by 1:1 (v/v)
PBS-methanol. Cells were fixed with methanol for ˜10 min and plates
were dried overnight. The cells were stained with 0.25% crystal violet
for 10 min, rinsed with tap water followed by distilled water. Cells were
dried at room temperature for ˜24 h. The stained cells were
solubilized in 3.5 mM of an aqueous SDS solution and ethanol (SDS added
first; followed by an equal volume ethanol). The OD was read at 560 nm.
FIG. 23 illustrates a typical result of the present experiment. From FIG.
24, it can be deduced that when the concentration of either isomer of 6b
is increased, the surviving fraction decreases at a constant rate.
Moreover, data in FIG. 24 suggests that 6b slow is more cytotoxic to U-87
cells compared to the 6b fast isomer.

Example 13

DNA Uptake of Compound 6b in U-87 Human Glioblastoma and the Subcellular
Distribution of Radioactivity Therein

[0185] U-87 cells were plated in T75 flasks and allowed to attach for 24
h. Compounds 6b-fast and 6b slow were added to cells at 1 μCi/mL (37
kBq/mL) and 5 μCi/mL (185 kBq/mL). Cells were incubated with
radioactive compounds from 24 h to 120 h. The radioactive medium was
removed. Cells were washed twice with fresh, nonradioactive medium. Cells
were trypsinized and their numbers and radioactive content determined. A
similar study was also conducted in 96- and 24-well formats, which
allowed more rapid analyses of several concentrations of 6b. Harvested
cells were processed using NE-PER nuclear and cytoplasmic extraction
reagents (Thermo Fisher Scientific, Rockford, Ill.). FIG. 25 shows the
uptake and subcellular distribution of both diastereomers of 6b expressed
in terms of the 125I radioactivity (cpm/cell) in cytoplasm, in
nucleus (i.e., DNA), and the total radioactivity in cell. U-87 cells
plated in T75 flasks and allowed to grow for 24 h. Cells were treated
with 0.75 Ci/mL 6b fast and 6b slow for 40 h at which time the
radioactive medium was removed and replaced with fresh media. Cells were
grown in fresh medium for additional 24 h and 72 h. Cells were
trypsinized and DNA was extracted using the Qiagen method (Qiagen
Genomic-tip 20/G; Qiagen, Valencia, Calif.). FIG. 26 shows the DNA
content expressed in cpm/cell. The experiment demonstrates that following
the uptake of the diastereomers into the cell, both isomers
preferentially reside in the nucleus. Moreover, 6b slow demonstrates a
greater overall cellular and nuclear uptake when compared to 6b fast.

[0186] U-87 cells were plated in 96-well plates and allowed to attach for
˜48 hours. The used medium was replaced with 6b-containing medium
and the cells were grown in the presence of radioactivity for 40 h. The
radioactive media was removed after 40 h of exposure. Aliquots (0.05 mL)
of the radioactive medium were counted in a gamma counter. Cell
monolayers were washed with PBS, PBS-methanol (2 min), and fixed in
methanol (10 min). Fixed cells were allowed to dry and were stained with
0.25% crystal violet (10 min). To each well 0.1 mL ethanol was added (2
h) followed by 0.1 mL 3.5 mM SDS in water. OD at 560 nm was read.
Solubilized cells were transferred into gamma counter tubes to determine
their radioactive content. The plate was divided into individual wells
and these were also added to the gamma counter tubes. FIG. 27 illustrates
the concentration-dependent uptake. The experiment demonstrates that of
the two diastereomers of 6b, 6b slow displayed almost a two-fold greater
uptake by U-87 human glioblastoma cells compared to 6b fast.

[0187] U-87 cells were plated for 24 h before treatment and then treated
for 24 h with concentrations of 6b fast and 6b slow ranging from 0.5
μCi/mL to 5 μCi/mL (18.5 to 185 kBq/mL). Cells were harvested,
washed, and their numbers counted using a Cellometer® cell counter.
Cells were diluted in fresh medium. Cells from each concentration of 6b
fast and 6b slow were plated in three T25 flasks at two densities, 100
cells/flask and 500 cells/flask. Control cells were sham-treated with PBS
and processed in a manner identical to cells treated with 6b. Cells were
periodically examined and fresh medium was added every 5-7 days. Three
weeks after plating, media was removed from the control flasks, cells
were washed with 5 mL ice-cold PBS, followed by 5 mL PBS/methanol (1:1;
v/v), and fixed in 5 mL methanol for 10 minutes. The methanol was
discarded and flasks were left open to dry for a few hours. Crystal
violet (5 mL; 0.25% in 1:1 PBS/methanol) was added to each flask to stain
the cells. After approximately 10 min, the dye was removed and flasks
were rinsed first with tap water followed by distilled water and were
left to dry. The 6b-treated cells were monitored for additional 4 weeks
during which time colonies were not formed in either 100 cells/flask or
500 cells/flask plating densities. This finding indicates the
extraordinarily effective killing of U-87 cells by 6b.

[0195] Four consecutive radioiodinations were carried out according to
General Procedure E within the 0.25-7.1 mCi range, to give overall 9.74
mCi of 12b. An average isolated yield of the product was 87%. The latest
radiolabeling was performed with the diastereomeric stannane 12a
(˜100 μg, ˜97% pure, UV at 280 nm) and [125I]NaI/NaOH
(65 μL, 7.1 mCi). The HPLC purification of the product proceeded on
ACE C18, 100 {circumflex over (Å)} (5 μm, 4.6×250 mm)
column; eluent: solvent A 10% MeCN in water, solvent B MeCN; eluted at
1.0 mL/min with a linear gradient of B from 0-40% over 40 min, followed
by a gradient of B from 40-95% for the period of 20 min. The
radioactivity peak of 12b (6.32 mCi, 89%) was collected in four
fractions, within 21-25 min after the injection of ˜500 μL (6.92
mCi) of the reaction mixture. An excess of the unreacted tin precursor
12a was separated from the radioiodinated product, eluting ˜5 min
later (tR=30.2 min). Combined fractions containing 12b were
evaporated, reconstituted in MeCN (˜1.8 mCi/mL) and 10 μL volume
(˜18 Ci) was re-injected on the HPLC: ACE C18, 100 {circumflex over
(Å)} (5 μm, 4.6×250 mm) column; eluent: solvent A 10% MeCN
in water, solvent B MeCN; eluted at 0.8 mL/min with a linear gradient of
B from 0-40% over 45 min, then a gradient of B from 40-95% for a period
of 15 min. Analysis showed a mixture (44:56 ratio) of diastereomers: 12b
fast, tR=26.4 min and 12b slow, tR=26.7 min (≧98% pure,
Bioscan NaI(T)). Diastereomers of 12b were not separated preparatively.
Co-injected solutions (in 50% MeCN in water) of the purified 12b and the
corresponding nonradioactive analog 12, with a parallel monitoring of the
radioactivity and UV signal, has shown the identical elution mobility of
both analogs. Diastereomer 12b fast eluted at tR=26.3 min and 12b
slow at tR=26.7 min, together with 12fast; tR=26.1 min and 12
slow; tR=26.4 min.

[0196] The total amount of prepared 13b was 12.5 mCi, obtained in five
successive radioiodinations of 13a, carried out within 0.25-5.1 mCi
range. Each reaction proceeded according to General Procedure E. An
average isolated yield of the product was 73%. The latest radiolabeling
was performed with the diastereomeric stannane 13a (˜100 μg) and
[125I]Na/NaOH (45 μL, 4.71 mCi). The HPLC purification proceeded
efficiently on Jupiter C18, 300 {circumflex over (Å)} (5 μm,
4.6×250 mm) column; eluent: solvent A 10% MeCN in water, solvent B
MeCN. A column was eluted at 1.0 mL/min with a linear gradient of B from
0-95% over 45 min, then 95% B for the period of 15 min. The radioactivity
peak of 13b (3.35 mCi, 71%) was collected within three fractions (24-27
min) after the injection of ˜300 μL (4.35 mCi) of the reaction
mixture. An excess of the unreacted tin precursor 13a was separated from
the radioiodinated product, eluting ˜6 min later (tR=29.6
min). Combined fractions containing the purified 13b were evaporated,
reconstituted in MeCN (˜1.2 mCi/mL) and 10 μL volume (˜12
Ci), was re-injected on the HPLC column: Jupiter C18, 300 {circumflex
over (Å)} (5 μm, 4.6×250 mm); eluent: solvent A 10% MeCN in
water, solvent B MeCN; eluted at 1.0 mL/min with a linear gradient of B
from 0-95% over 45 min, then 95% B for the period of 15 min. The analysis
showed a mixture (47:53 ratio) of diastereomers 13b fast, tR=23.4
min and 13b slow, tR=23.7 min (≧98% pure, Bioscan NaI(T)).
Diastereomers of 13b were not separated preparatively. Co-injected
solutions (in 50% MeCN) of the purified 13b (˜18 μCi, 15 μL)
and the nonradioactive analog 13 (˜17 μg, 25 μL) were
analyzed, with monitoring the radioactivity (Bioscan NaI(T)) and UV at
280 nm, on ACE C18, 100 {circumflex over (Å)} (5 μm, 4.6×250
mm) column; eluent: solvent A 10% MeCN in water, solvent B MeCN; eluted
at 1.0 mL/min with a linear gradient of B from 0-95% over a period of 60
min. The analysis confirmed the identical elution of both: [125I]-
and [127I]-iodoanalogs.

[0197] General Procedure E was carried out within 0.5-5.7 mCi range, to
give after four conducted radioiodinations, 12.6 mCi of 14b in an average
yield of 86%. The latest radiolabeling proceeded with stannane 14a
(˜110 μg) and [125I]NaI/NaOH (60 μL, 5.71 mCi). The HPLC
purification of the crude product was achieved on Jupiter C18, 300
{circumflex over (Å)} (5 μm, 4.6×250 mm) column; eluent:
solvent A 40% MeCN in water, solvent B MeCN. A column was eluted at 1.0
mL/min the flow rate, with a linear gradient of B from 0-95% over 35 min,
followed by 95% B for the period of 25 min. The product (5.02 mCi, 88%)
collected within 21-23 min after the injection of 425 μL (˜5.5
mCi) of the reaction mixture was separated from an excess of the
unreacted tin precursor 14a, which eluted ˜10 min later (32.8-33.6
min). Appropriate fractions were combined, evaporated with a stream of
nitrogen, and the residue further dried in high vacuum. The mixture of
purified 14b (˜12 μCi, μL) and its corresponding
nonradioactive analog 14 (˜15 μg, 20 μL) in acetonitrile, was
reinjected onto the HPLC, using the same settings as during the
separation of the crude product. The analysis has shown 14b fast at
tR=21.2 min and 14b slow; tR=21.7 min, co-eluting with
diastereomers of the [127I]-iodoanalog: 14 fast, tR=20.9 min
and 14 slow, tR=21.6 min. Diastereomers of 14b were most efficiently
separated on ACE C18, 100 {circumflex over (Å)} (5 μm,
4.6×250 mm) column, eluted with 50% MeCN in water at the 0.8 mL/min
flow rate. Each isomer, 14b fast (tR=41.2 min) and 14b slow
(tR=45.7 min), was ≧98% pure (Bioscan NaI(T) detector). The
complete separation of diastereomers in the single HPLC run was attained,
if the total amount of 14b loaded onto a column was ≦320 μCi.
Larger lots of single diastereomers were obtained in multiple injections.

[0198] The examples provided, above, indicate that compounds of formula
(I) bind to a malignant tumor cell marker, BChE, access the nucleus,
incorporate into the DNA of a tumor cell, and selectively deliver a
radioisotopically labeled moiety that kills malignant tumors. Compounds
of formula I can also be imaged in xenografted tumors in mice after
delivery, thereby allowing for diagnostic studies of a cancer in vivo.
Additionally, of the diastereomers of formula I that were tested, the
slow eluting isomers are the more active therapeutics.

[0199] Several of the compounds described herein have exhibited activity
for the treatment and diagnosis of Alzheimer's disease. See U.S. Patent
Publication No. 2009/0117041, which is commonly owned with the present
application.

[0200] A number of patent documents and non-patent documents are cited in
the foregoing specification in order to describe the state of the art to
which this invention pertains. The entire disclosure of each of the cited
documents is incorporated by reference herein.

[0201] While various embodiments of the present invention have been
described and/or exemplified above, numerous other embodiments will be
apparent to those skilled in the art upon review of the foregoing
disclosure. The present invention is, therefore, not limited to the
particular embodiments described and/or exemplified, but is capable of
considerable variation and modification without departure from the scope
of the appended claims.

[0202] Furthermore, the transitional terms "comprising", "consisting
essentially of" and "consisting of", when used in the appended claims, in
original and amended form, define the claim scope with respect to what
unrecited additional claim elements or steps, if any, are excluded from
the scope of the claim(s). The term "comprising" is intended to be
inclusive or open-ended and does not exclude any additional, unrecited
element, method, step or material. The term "consisting of" excludes any
element, step or material other than those specified in the claim and, in
the latter instance, impurities ordinary associated with the specified
material(s). The term "consisting essentially of" limits the scope of a
claim to the specified elements, steps or material(s) and those that do
not materially affect the basic and novel characteristic(s) of the
claimed invention. All resonant sensors and methods of use thereof that
embody the present invention can, in alternate embodiments, be more
specifically defined by any of the transitional terms "comprising",
"consisting essentially of" and "consisting of".